Anti-hepcidin antibodies and uses thereof

ABSTRACT

Monoclonal antibodies are provided that selectively bind human hepcidin-25 and are characterized as having high affinity for human hepcidin-25 and strong human mature hepcidin neutralizing properties. The antibodies of the invention are useful therapeutically for increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit in a human and for the treatment and diagnosis of mature hepcidin-promoted disorders such as anemia, in a human subject.

The present invention is in the field of medicine, particularly in thefield of antibodies against human mature hepcidin. More specifically,the invention relates to hepcidin-25 selective monoclonal antibodieswhich are capable of neutralizing human mature hepcidin bioactivity and,therefore, are useful for increasing serum iron levels, reticulocytecount, red blood cell count, hemoglobin, and/or hematocrit in a humanfor purposes of treating or preventing a human mature hepcidin-promoteddisease, condition, or disorder such as anemia.

Presently, suitable and effective therapies for anemia, or for anemia ofchronic disease, are limited. Specifically, erythropoietinadministration is effective in only about 50% of all the patients, andis associated with undesirable side effects. Furthermore, transfusionsare undesirable due to the risks of contamination, infection, and ironoverload.

Human hepcidin, a polypeptide expressed predominantly by hepatocytes, isbelieved to be an important iron-regulating protein that negativelyregulates intestinal iron absorption, iron recycling by macrophages, andiron mobilization from hepatic iron stores. Overproduction of hepcidinappears to play a primary role in the pathophysiology of anemia and/oranemia of chronic disease.

Human hepcidin is encoded as an 84-amino acid prepropeptide containing atypical N-terminal 24-amino acid endoplasmic reticulum-targeting signalsequence, and a 35-amino acid proregion with a consensus furin-cleavagesite immediately followed by the C-terminal 25 amino acid bioactiveiron-regulatory hormone, human hepcidin-25 (SEQ ID NO: 1). VariousN-terminal truncated forms of human hepcidin-25, such as humanhepcidin-20 (i.e., amino acids 6-25 of SEQ ID NO: 1) and humanhepcidin-22 (amino acids 4-25 of SEQ ID NO: 1) are also known to form invivo.

Although antibodies to human hepcidin have been reported previously(see, e.g., U.S. Patent Application Publications 2004/0096990 and2007/0224186, and PCT International Patent Application Publication WO2008/097461), there still remains a great need in the art for additionaldrugs to treat diseases and disorders associated with anemia includinganemia of chronic disease such as anemia of cancer and anemia ofinflammation. Because hepcidin-25 is the most, if not the only,physiologically relevant form of hepcidin in humans, antibodies thatselectively target human hepcidin-25 as compared to hepcidinpolypeptides that are not physiologically relevant, are particularlyneeded. Therefore, the present invention provides selective, highaffinity, engineered therapeutic antibodies against human hepcidin-25that have numerous advantages in the treatment or diagnosis of disordersassociated with elevated levels of mature hepcidin such as anemia. Forexample, these antibodies, being high affinity, neutralizing, humanengineered and highly selective for physiologically relevant forms ofhepcidin in humans, will reduce the risk for side-effects and theclinical dose and frequency of dosing required for effective treatment.The present invention also includes preferred nucleic acids encodingpreferred selective hepcidin-25 antibodies wherein the nucleic acidshave been engineered to remove cryptic splice sites that result inundesirable aggregation of certain antibodies of the invention uponexpression by mammalian host cells. Thus, additional benefits derivedfrom this the present invention include improved yield of the antibodyproduct of a desirable degree of purity, thereby cutting costs ofproduction, as well as a greater degree of clinical effectiveness andsafety for the antibody product administered.

Furthermore, commercially available immunoassays for human hepcidin donot differentiate the active, physiologically relevant forms of humanhepcidin from inactive, physiologically non-relevant hepcidin species(see, for example, Kemna, E. H., et al., Haematologica, 93(1):90-7(2008)). Presently, most methods to selectively assay for hepcidin-25involve LC/MS (liquid chromatography/mass spectroscopy) or similarcumbersome methods which require the separation of the various forms ofhepcidin (see, for example, (Gutierrez, J. A., et al., BioTechniques,38:S13-S17 (2005), Murphy, et al., Blood, 110: 1048-54 (2007) and Kemna,E. H., et al., Clin. Chem. 53:620-8 (2007)). While these assays may beaccurate and precise, their complexity, expense, and the high level ofoperator expertise required inhibit their routine implementation.Accordingly, there is also a great need for additional antibodies thatbind human mature hepcidin with high affinity for their application inrelatively simple, rapid, and robust immunoassay for the specificdetection or measurement of mature forms of human hepcidin fordiagnostic and/or prognostic applications.

The present invention provides antibodies that bind human hepcidin-25with a binding affinity (K_(D)) of about 800 pM or less as determined bysurface plasmon resonance (SPR) at 25° C. Preferably, the antibody has adissociation rate (k_(off)) for human hepcidin-25 between about 8.5×10⁻³s⁻¹ and about 1.8×10⁻⁴ s⁻¹ as determined by SPR at 25° C. Morepreferably, the antibody has a K_(D) for human hepcidin-25 between about400 pM to about 30 pM as determined by SPR at 25° C. Even morepreferably, the antibody has a K_(D) for human hepcidin-25 between about200 pM to about 30 pM as determined by SPR at 25° C. Even morepreferably, the antibody has an IC₅₀ between about 100 nM and about 25nM in an in vivo assay of hepcidin-25 bioactivity, preferably, whereinthe assay measures an IL-6-induced decrease in serum iron levels. Evenmore preferably, the antibody has an IC₅₀ between about 100 nM and about50 nM in an in vitro assay of hepcidin-25 bioactivity, preferably,wherein the assay measures hepcidin-induced internalization and/ordegradation of ferroportin. Even more preferably, the antibodiescomprise at least one of the CDRs selected from the group consisting ofi) a HCDR3 having the amino acid sequence as shown in SEQ ID NO: 75, andii) a LCDR3 having the amino acid sequence as shown in SEQ ID NO: 62.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 800 pM or less and comprises a heavychain variable region (“HCVR”) polypeptide and a light chain variableregion (“LCVR”) polypeptide wherein (i) the HCVR and the LCVRpolypeptides have the amino acid sequences as shown in SEQ ID NOs: 148and 126, respectively; (ii) the HCVR and the LCVR polypeptides have theamino acid sequences as shown in SEQ ID NOs: 128 and 127, respectively;(iii) the HCVR and the LCVR polypeptides have the amino acid sequencesas shown in SEQ ID NOs: 151 and 125, respectively; or (iv) the HCVR andthe LCVR polypeptides have the amino acid sequences as shown in SEQ IDNOs: 150 and 124, respectively.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 800 pM or less and comprises a heavychain polypeptide and a light chain polypeptide wherein (i) the heavychain and light chain polypeptides have the amino acid sequences asshown in SEQ ID NOs: 6 and 14, respectively; (ii) the heavy chain andlight chain polypeptides have the amino acid sequences as shown in SEQID NOs: 7 and 15, respectively; (iii) the heavy chain and light chainpolypeptides have the amino acid sequences as shown in SEQ ID NOs: 9 and17, respectively; or (iv) the heavy chain and light chain polypeptideshave the amino acid sequences as shown in SEQ ID NOs: 8 and 16,respectively.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 800 pM or less and comprises a LCVRpolypeptide comprising 3 CDR sequences which are present together in aFab listed in Table 1 herein and which are present in the antibody inthe same CDR position as shown in Table 1. Preferably, such an antibodycomprises a LCVR polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 101-127.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 800 pM or less and comprises a HCVRpolypeptide comprising 3 CDR sequences which are present together in aFab listed in Table 2 herein and which are present in the antibody inthe same CDR position as shown in Table 2. Preferably, such an antibodycomprises a HCVR polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 128-151.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 800 pM or less and comprises i) a LCVRpolypeptide comprising 3 CDRs which are present together in a Fab listedin Table 1 and which are present in the antibody in the same CDRposition as shown in Table 1, and ii) a HCVR polypeptide comprising 3CDRs which are present together in a Fab listed in Table 2 and which arepresent in the antibody of the invention in the same CDR position asshown in Table 2. Preferably, such an antibody comprises 6 CDRs whichare present together in a Fab listed in Table 3 herein and which arepresent in the antibody in the same CDR position as shown in Table 3.

The present invention includes an antibody that selectively binds humanhepcidin-25 with a K_(D) of about 200 pM or less and comprises (i) aLCVR polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 101-127, and (ii) a HCVR polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NOs:128-151.

The present invention also includes an antibody that selectively bindshuman hepcidin-25 with a K_(D) of about 200 pM or less and comprises twoheavy chain polypeptides and two light chain polypeptide, and whereineach of the heavy chain polypeptides have the amino acid sequence asshown in SEQ ID NO: 8 and each of the light chain polypeptides have theamino acid sequence as shown in SEQ ID NO: 16.

In other aspects, the invention provides isolated nucleic acid moleculesencoding antibodies of the invention; vectors comprising nucleic acidmolecules encoding antibodies of the invention, optionally,operably-linked to control sequences recognized by a host celltransformed with the vector; host cells comprising vectors comprisingnucleic acid molecules encoding antibodies of the invention; a processfor producing an antibody of the invention comprising culturing hostcells comprising vectors comprising nucleic acid molecules encodingantibodies of the invention so that the nucleic acid is expressed and,optionally, recovering the antibody from the host cell culture medium.

In another aspect, the invention provides a pharmaceutical compositioncomprising an antibody of the invention and a pharmaceuticallyacceptable carrier or diluent. Preferably, the pharmaceuticalcomposition comprises a homogeneous or substantially homogeneouspopulation of an antibody of the invention and a pharmaceuticallyacceptable carrier or diluent.

In another embodiment, the invention provides an antibody thatselectively binds human hepcidin-25 with a K_(D) of about 800 pM or lessfor use in therapy. The invention also provides an antibody thatselectively binds human hepcidin-25 with a K_(D) of about 800 pM or lessfor use in treating or preventing anemia in a subject.

The present invention includes the use of an antibody that selectivelybinds human hepcidin-25 with a K_(D) of about 800 pM or less for thepreparation of a medicament for the treatment of anemia, includinganemia of chronic disease and anemia of cancer. The invention furtherincludes the use of an antibody that selectively binds human hepcidin-25with a K_(D) of about 800 pM or less for the preparation of a medicamentfor increasing serum iron levels, reticulocyte count, red blood cellcount, hemoglobin, and/or hematocrit in an animal, preferably amammalian species, more preferably a human subject.

The present invention includes a method of increasing serum iron levels,reticulocyte count, red blood cell count, hemoglobin, and/or hematocritthat comprises administering to a human subject in need thereof, aneffective amount of an antibody that selectively binds human hepcidin-25with a K_(D) of about 800 pM or less.

In another aspect, the invention provides a method for treating in apatient a mature hepcidin-promoted disorder which benefits from anincrease in serum iron levels, reticulocyte count, red blood cell count,hemoglobin, and/or hematocrit, including, but not limited to, anemia,e.g., anemia resulting from infection, inflammation, chronic disease,and/or cancer wherein said method comprises administering an effectiveamount of a hepcidin-25 selective antibody of the invention to a patientin need thereof.

The present invention further provides an immunoassay selective forhuman mature hepcidin. The method includes: first, obtaining a sample tobe assayed for human mature hepcidin and contacting the sample with anantibody of the invention under suitable conditions for binding andallowing any human mature hepcidin present to form an antigen-antibodycomplex; then detecting the presence or absence of the complex; and/ordetermining the amount of the complex in the sample by an immunoassaymethod.

The present invention further provides methods of diagnosing a humanmature hepcidin-promoted condition in a patient by determining the levelof human mature hepcidin in a sample of a biological fluid from thepatient and comparing the level of human mature hepcidin in the samplewith the level of human mature hepcidin in a sample of biological fluidfrom one or more control individuals or with a reference standard.

A method of monitoring a mature hepcidin-promoted disorder in a patientis also provided. The method includes determining the level of maturehepcidin in a sample of a biological fluid from a patient sufferingfrom, or at risk of, a mature hepcidin-promoted disorder at a first timepoint; determining the level of mature hepcidin in one or more samplesof the biological fluid from the patient at one or more different timepoints; comparing the levels of mature hepcidin determined at differenttime points and thereby monitoring the mature hepcidin-promoteddisorder. The invention further provides a kit for performing animmunoassay, including an antibody of the invention and a suitablecontainer.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts a MALDI-TOF mass spectrum of multiple forms of humanhepcidin isolated from human sera. Signal 1 has a mass which isconsistent with the expected mass of intact human hepcidin-25. Signals2, 3, and 4 have masses that are consistent with N-terminally truncatedforms of human mature hepcidin (hepcidin-24, hepcidin-22, andhepcidin-20). The mass spectrum was generated on a MALDI-TOF massspectrometer utilizing a positive ion, linear mode method witha-cyano-4-hydroxycinnamic acid (peptide matrix) as sample matrix asdescribed in Example 5 below.

FIG. 2 depicts a MALDI-TOF mass spectrum of the same sample as in FIG. 1using 3,5-dimethyl-4 hydroxycinnamic acid (sinapinic acid matrix) assample matrix. Signal 1 represents intact human hepcidin-25. No signalfor pro-hepcidin was observed. The mass spectrum was generated on a massspectrometer utilizing a positive ion, linear mode method as describedin Example 5 below.

FIG. 3A shows the amino acid sequences of fully human light chainframework O2 with interspersed CDRs. The four framework regions arelabeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 39, 40, 96, and 97,respectively).

FIG. 3B shows the amino acid sequence of the human heavy chain frameworkVH1-69 with interspersed CDRs. The four framework regions are labeledFRH1-4 (SEQ ID NOs: 35-38, respectively).

FIG. 4A shows the amino acid sequences of the human light chainframework O18 with interspersed CDRs The four framework regions arelabeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 154, 40, 156, and 97,respectively).

FIG. 4B shows the amino acid sequence of the human heavy chain frameworkVH1-18 with interspersed CDRs. The four framework regions are labeledFRH1, 2, 3, and 4 (SEQ ID NOs: 157, 36, 158, and 38, respectively).

FIG. 5A shows the amino acid sequences of the human light chainframework L12 with interspersed CDRs The four framework regions arelabeled as FRL1, 2, 3, and 4 (SEQ ID NOs: 159, 40, 160, and 97,respectively).

FIG. 5B shows the amino acid sequence of the human heavy chain frameworkVH1-46 with interspersed CDRs. The four framework regions are labeledFRH1, 2, 3, and 4 (SEQ ID NOs: 157, 36, 161, and 38, respectively).

The following abbreviations are used herein: ACN: acetonitrile, BSA:bovine serum albumin, DTT: dithiothreitol, EDTA: ethylenediaminetetraacetic acid, ELISA: enzyme linked immunosorbent assay, IMAC:immobilized metal-affinity chromatography, IPTG: isopropylβ-D-1-thiogalactopyranoside, Mab: monoclonal antibody, Mabs: monoclonalantibodies, MALDI-TOF: Matrix-Associated Laser DesorptionIonization-Time of Flight, PBS: phosphate-buffered saline, SPR: surfaceplasmon resonance, TFA: trifluoroacetic acid. All amino acidabbreviations used in this disclosure are those accepted by the UnitedStates Patent and Trademark Office as set forth in 37 C.F.R. § 1.822(B)(2).

When used herein, the term “hepcidin” refers to any form of the hepcidinprotein known to be present in mammals. When used herein, the term“mature hepcidin” refers to any mature, bioactive form of the hepcidinprotein expressed in mammals. When used herein, the phrase “humanhepcidin” refers to any form of the hepcidin protein present in humans.When used herein, the phrase “human hepcidin-25” refers to the matureform of human hepcidin having the amino acid sequence as shown in SEQ IDNO: 1.

The term “antibody” in reference to an anti-hepcidin antibody of theinvention (or simply, “antibody of the invention”), as used herein,refers to a human engineered monoclonal antibody or a fully humanmonoclonal antibody, unless otherwise indicated. Preferably, theantibodies of the invention are human engineered antibodies. Antibodiesof the invention can be produced using e.g., recombinant technologies,phage display technologies, synthetic technologies, e.g., CDR-grafting,or combinations of such technologies or other technologies readily knownin the art. “Monoclonal antibody” refers to an antibody that is derivedfrom a single copy or clone, including e.g., any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.An antibody as used herein can be an intact antibody (comprising acomplete or full length Fc region), or a portion or fragment of anantibody comprising an antigen-binding portion, e.g., a Fab fragment,Fab′ fragment, or F(ab′)₂ fragment of a human engineered or fully humanantibody. Preferred antigen-binding fragments of an antibody of theinvention retain the ability to inhibit or neutralize one or morebioactivities characteristic of a mature form of a mammalian hepcidin invivo or in vitro. For example, in one embodiment, an antigen-bindingportion of an antibody of the invention can inhibit the interaction ofhuman hepcidin-25 with one or more receptors, e.g., human ferroportin(SEQ ID NO: 25), and/or can inhibit hepcidin-induced internalization offerroportin.

Furthermore, an “antibody of the invention” or simply “antibody” as usedherein can be a single chain Fv fragment that may be produced by joiningthe DNA encoding the LCVR and HCVR with a linker sequence. (See,Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp 269-315, 1994).It is understood that regardless of whether antigen-binding fragments orportions are specified, the term “antibody” as used herein includes suchfragments or portions as well as single chain forms, unless indicatedotherwise.

The terms “selective” or “selectively” used herein in reference to ananti-hepcidin-25 antibody or the binding thereof, respectively, refersto an antibody that selectively binds hepcidin-25 with a K_(D) of about1000-, 500-, 200-, 100-, 50-, 10-, or about 5-fold lower than theantibody binds at least one precursor form of hepcidin-25 and/or atleast one N-terminally truncated species of mature hepcidin known toform in the same mammalian species, as measured by SPR at 25° C.Additionally, or alternatively, a hepcidin-25 selective antibody of theinvention binds to hepcidin-25 but does not bind, or minimally binds, toat least one precursor form of hepcidin-25 and/or at least oneN-terminally truncated species of mature hepcidin known to form in thesame mammalian species as determined by immunoassay and/or MALDI-TOFmass spectrometry methods used by those skilled in the art including,but not limited to, the assay described in Example 5 herein. Preferably,an anti-hepcidin-25 selective antibody of the present invention bindshuman hepcidin-25 with a K_(D) of about 1000-, 500-, 200-, 100-, 50-,10-, or about 5-fold lower than the antibody binds human pro-hepcidin,preferably, human pro-hepcidin having the amino acid sequence shown inSEQ ID NO: 34, and/or at least one N-terminally truncated form of humanmature hepcidin, as measured by SPR at 25° C. Additionally, oralternatively, an anti-hepcidin-25 selective antibody of the inventionbinds to human hepcidin-25 but does not bind, or minimally binds, tohuman pro-hepcidin, preferably, the human pro-hepcidin having the aminoacid sequence shown in SEQ ID NO: 34, and/or at least one N-terminallytruncated species of mature hepcidin known to form in the same mammalianspecies as determined by immunoassay and/or MALDI-TOF mass spectrometrymethods used by those skilled in the art including, but not limited to,the assay described in Example 5 herein. More preferably, ananti-hepcidin-25 selective antibody of the present invention binds humanhepcidin-25 with a K_(D) of about 1000-, 500-, 200-, 100-, 50-, 10-, orabout 5-fold lower than the antibody binds human pro-hepcidin having theamino acid sequence shown in SEQ ID NO: 34 and at least one N-terminallytruncated form of human mature hepcidin, as measured by SPR at 25° C.Additionally, or alternatively, an anti-hepcidin-25 selective antibodyof the invention binds to human hepcidin-25 but does not bind, orminimally binds, to human pro-hepcidin (SEQ ID NO: 34), and at least oneN-terminally truncated species of mature hepcidin known to form in thesame mammalian species as determined by immunoassay and/or MALDI-TOFmass spectrometry methods used by those skilled in the art including,but not limited to, the assay described in Example 5 herein. Mostpreferably, an anti-hepcidin-25 selective antibody of the presentinvention i) binds human hepcidin-25 with a K_(D) of about 1000-, 500-,200-, 100-, 50-, 10-, or about 5-fold lower than the antibody bindshuman pro-hepcidin (SEQ ID NO: 34) and ii) binds human hepcidin-25 witha K_(D) of about 1000-, 500-, 200-, 100-, 50-, 10-, or about 5-foldlower than the antibody binds human hepcidin-20 (i.e., amino acids 6-25of SEQ ID NO: 1) and/or human hepcidin-22 (amino acids 4-25 of SEQ IDNO: 1), as measured by SPR at 25° C. Additionally, or alternatively, ananti-hepcidin-25 selective antibody of the invention binds to humanhepcidin-25 but does not bind, or minimally binds, to i) humanpro-hepcidin (SEQ ID NO: 34) and ii) human hepcidin-20 and/or humanhepcidin-22, as determined by immunoassay and/or MALDI-TOF massspectrometry methods used by those skilled in the art including, but notlimited to, the assay described in Example 5 herein.

The term “detect” or “detecting” is used in the broadest sense toinclude quantitative, semi-quantitative or qualitative measurements of atarget molecule. In one aspect, methods described herein may onlydetermine the presence or absence of a particular hepcidin polypeptidein a biological sample and, thus, that the hepcidin polypeptide isdetectable or, alternatively, undetectable in the sample as determinedby the method.

The term “epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody at one or more of the antibody'santigen-binding regions.

The term “bioactivity,” in reference to an antibody of the invention,includes, but is not limited to, epitope or antigen binding affinity,the in vivo and/or in vitro stability of the antibody, the immunogenicproperties of the antibody, e.g., when administered to a human subject,and/or the ability to neutralize or antagonize a bioactivity ofhepcidin, in vivo or in vitro, including, but not limited to, inhibitionof serum iron level dysregulation in an inflammation, e.g., IL-6,challenge assay, e.g., as described in Example 4 herein. Theaforementioned properties or characteristics can be observed or measuredusing art-recognized techniques including, but not limited to,scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN),fluorescence quenching, fluorescence ELISA, competitive ELISA, SPRanalysis including, but not limited to, SPR analysis using a BIAcorebiosenser, in vitro and in vivo neutralization assays without limit(see, for example, International Publication No. WO 2006/062685),receptor binding, and immunohistochemistry with tissue sections fromdifferent sources including human, primate, or any other source as theneed may be.

The term “bioactivity” in reference to mature hepcidin includinghepcidin-25 includes, but is not limited to, specific binding of maturehepcidin to another protein including, but not limited to, its receptorferroportin, one or more ferroportin-mediated functions of maturehepcidin, such as mature hepcidin-induced internalization and/ordegradation of ferroportin (see, e.g., Nemeth, E., et al., HepcidinRegulates Iron Efflux by Binding to Ferroportin and Inducing ItsInternalization, Science 306, 2090-2093, (2004)), mature hepcidinregulation of ferroportin-mediated iron efflux, serum iron levels,reticulocyte count, red blood cell count, hemoglobin, and/or hematocritin a human, protein stability, i.e., mature hepcidin affecting thelevels or activity of another protein in vivo or in vitro, and hepcidinexpression levels and/or tissue distribution.

The term “inhibit” or “neutralize” as used herein with respect to abioactivity of an antibody of the invention means the ability tosubstantially antagonize, prohibit, prevent, restrain, slow, disrupt,eliminate, stop, reduce or reverse a bioactivity of human maturehepcidin, including, but not limited to, a human mature hepcidinbioactivity as measured in Example 3 or 4 herein.

The antibodies of the present invention are characterized by having aK_(D) for human hepcidin-25 less than about 1000 pM, preferably, lessthan about 800 pM, more preferably, less than about 400 pM, even morepreferably, less than about 200 pM, even more preferably, less thanabout 100 pM, even more preferably, less than about 75 pM, or mostpreferably, less than about 50 pM, as determined by SPR at 25° C.Preferably, the antibody selectively binds human hepcidin-25 with aK_(D) less than about 800 pM, preferably, less than about 400 pM, morepreferably, less than about 200 pM, even more preferably, less thanabout 100 pM, even more preferably, less than about 75 pM, or mostpreferably, less than about 50 pM, as determined by SPR at 25° C. alsoselectively binds at least one hepcidin-25 of another species such ascynomolgous monkey hepcidin-25. More preferably, such antibodiesselectively bind cynomolgous monkey hepcidin-25 with a K_(D) less thanabout 800 pM, even more preferably, less than about 400 pM, even morepreferably, less than about 200 pM, even more preferably, less thanabout 100 pM, even more preferably, less than about 75 pM, or mostpreferably, less than about 50 pM, as determined by SPR at 25° C.

The antibodies of the present invention include anti-hepcidin-25selective antibodies having a K_(D) for human hepcidin-25 between about800 pM and about 30 pM, preferably, between about 400 pM and about 30pM, more preferably, between about 200 pM and about 30 pM, even morepreferably, between about 100 pM and about 30 pM, even more preferably,between about 75 pM and about 30 pM, or most preferably, between about50 pM and about 30 pM, as determined by SPR at 25° C. Preferably, suchantibodies also have a K_(D) for cynomolgus monkey hepcidin-25 betweenabout 800 pM and 10 pM, more preferably, between about 400 pM and about10 pM, even more preferably, between about 200 pM and about 10 pM, evenmore preferably, between about 100 pM and about 10 pM, even morepreferably, between about 75 pM and 10 pM, or most preferably, betweenabout 50 pM and 10 pM, as determined by SPR at 25° C.

The antibodies of the present invention also include antibodies having aK_(D) for human hepcidin-25 and/or cynomolgus monkey hepcidin-25 whichis at least about 20-fold, at least about 30-fold, at least about40-fold, at least about 50-fold, at least about 60-fold, at least about70-fold, at least about 80-fold, at least about 90-fold, at least about100-fold, or at least 200-fold less than the antibody's K_(D) for mousehepcidin-25 and/or rat hepcidin-25, as determined by SPR at 25° C.

The antibodies of the present invention also include antibodies having ak_(off) rate from human hepcidin-25 between about 1×10⁻² s⁻¹ and about1.8×10⁻⁴ s⁻¹ preferably, between about 8.5×10⁻³ s⁻¹ and about 1.8×10⁻⁴s⁻¹ more preferably, between about 7.7×10⁻⁴ s⁻¹ and about 1.8×10⁻⁴ s⁻¹even more preferably, between about 6.5×10⁻⁴ s⁻¹ and about 1.8×10⁻⁴ s⁻¹or most preferably, between about 5.5×10⁻⁴ s⁻¹ and about 2.0×10⁻⁴ s⁻¹ asdetermined by SPR at 25° C. Preferably, such an antibody alsoselectively binds human hepcidin-25 with a K_(D) between about 800 pMand 30 pM, even more preferably, between about 400 pM and about 30 pM,even more preferably, between about 200 pM and about 30 pM, even morepreferably, between about 100 pM and about 30 pM, even more preferably,between about 75 pM and 50 pM, or most preferably, between about 50 pMand 30 pM, as determined by SPR at 25° C.

The present invention also includes antibodies that bind humanhepcidin-25, preferably, selectively, with a K_(D) of about 200 pM orless and neutralizes or antagonizes at least one human mature hepcidinbioactivity in vitro or in vivo. Preferably, an antibody of theinvention has an IC₅₀ lower than about 200 nM, more preferably, lowerthan about 100 nM, even more preferably, lower than about 75 nM, or mostpreferably, lower than about 50 nM in an in vitro or in vivo assay ofmature hepcidin bioactivity as described, for example, in Example 3 or 4herein.

An antibody of the invention also includes anti-human hepcidin-25binding, preferably, selectively binding, antibodies that significantlyinhibit IL-6-induced serum iron decreases in a cynomolgus monkey assayas described, for example, in Example 4 herein. Preferably, suchantibodies inhibit serum iron decreases in a cynomolgus monkey inducedby a 5 μg/kg dose of human IL-6 by at least about 30%, at least about40%, at least about by 50%, at least about by 60%, at least about 70%,at least about 80%, at least about by 90%, or at least about by 100%within about 6 hours of receiving an intravenous dose of the antibody atabout 10 mg/kg.

An antibody of the invention has an IC₅₀ lower than about 200 nM,preferably, less than about 100 nM, more preferably, less than about 75nM, even more preferably, less than about 50 nM, or most preferably,less than about 25 nM in the mature hepcidin-induced ferroportininternalization assay described in Example 3 herein. Preferably, anantibody of the invention has an IC₅₀ between about 200 nM and about 25nM, more preferably, between about 100 nM and about 50 nM, morepreferably, between about 100 nM and about 25 nM, even more preferably,between about 75 nM and about 25 nM, or most preferably, between about75 nM and about 50 nM in the mature hepcidin-induced ferroportininternalization assay described in Example 3 herein.

In another embodiment, an antibody of the invention has an IC₅₀ betweenabout 200 nM and about 25 nM, preferably, between about 100 nM and about50 nM, more preferably, between about 100 nM and about 25 nM, even morepreferably, between about 75 nM and about 25 nM, or most preferably,between about 75 nM and about 50 nM in the hepcidin-induced ferroportininternalization assay described in Example 3, a k_(off) rate from humanhepcidin-25 between about 1×10⁻² s⁻¹ and about 1.8×10⁻⁴ s⁻¹ preferably,between about 8.5×10⁻³ s⁻¹ and about 1.8×10⁻⁴ s⁻¹ more preferably,between about 7.7×10⁻⁴ s⁻¹ and about 1.8×10⁻⁴ s⁻¹ even more preferably,between about 6.5×10⁻⁴ s⁻¹ and about 1.8×10⁻⁴ s⁻¹, or most preferably,between about 5.5×10⁻⁴ s⁻¹ and about 2.0×10⁻⁴ s⁻¹, as determined by SPRat 25° C., and selectively binds human hepcidin-25 with a K_(D) betweenabout 800 pM and 30 pM, preferably, between about 400 pM and about 30pM, more preferably, between about 200 pM and about 30 pM, even morepreferably, between about 100 pM and about 30 pM, even more preferably,between about 75 pM and 50 pM, or most preferably, between about 50 pMand 30 pM, as determined by SPR at 25° C.

The term “Kabat numbering” as used herein is recognized in the art andrefers to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain regions of an antibody (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971); Kabat, et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242 (1991)).

A polynucleotide is “operably-linked” when it is placed into afunctional relationship with another polynucleotide. For example, apromoter or enhancer is operably-linked to a coding sequence if itaffects the transcription of the sequence.

The terms “subject” and “patient” used interchangeably herein, refer toa mammal, preferably, a human. In certain embodiments, the patient has adisorder that would benefit from a decreased level of hepcidin-25, adecrease in hepcidin-25 bioactivity, and/or an increase in serum ironlevel, reticulocyte count, red blood cell count, hemoglobin, and/orhematocrit,

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been operably-linkedincluding, but not limited to, plasmids and viral vectors. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced while other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and thereby,are replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperably-linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply “expression vectors”). Exemplary vectorsare well known in the art.

As used herein, the expressions “cell” and “host cell” are usedinterchangeably and refer to any prokaryotic cell (e.g., bacterial cellssuch as E. coli) or, preferably, eukaryotic cell (e.g., yeast cells,plant cells, insect cells, or mammalian cells such as CHO cells) whetherlocated in vitro or in vivo. A host cell includes cells transformed,transduced, transfected, or infected with one or more recombinantexpression vectors comprising a polynucleotide encoding an antibody ofthe invention. A host cell may be located in vitro or in vivo. Forexample, host cells may be located in a transgenic animal or atransgenic plant.

Each heavy chain of a full-length antibody is comprised of an N-terminalheavy chain variable region (herein “HCVR”) and a C-terminal heavy chainconstant region. Each light chain of a full-length antibody is comprisedof an N-terminal light chain variable region (herein “LCVR”) and aC-terminal light chain constant region. The HCVRs and LCVRs can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (“CDRs”), interspersed with regionsthat are more conserved, termed framework regions (“FR”). The functionalability of an antibody to bind a particular antigen or epitope islargely influenced by the six CDRs present in the variable region of theantibody. Each HCVR and LCVR is composed of three CDRs (HCDR1, HCDR2 andHCDR3 in the HCVR and LCDR1, LCDR2 and LCDR3 in the LCVR) and fourframework regions, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Accordingly, the term “CDR” or “complementarity determining region” asused herein, is intended to mean the non-contiguous antigen combiningsites found within the variable region of both heavy and light chainpolypeptides. These particular regions have been described by Kabat, etal., J. Biol. Chem. 252, 6609-6616 (1977), Kabat, et al., Sequences ofprotein of immunological interest, (1991), and by Chothia, et al., J.Mol. Biol. 196:901-917 (1987) and by MacCallum, et al., J. Mol. Biol.,262:732-745 (1996) where the definitions include overlapping or subsetsof amino acid residues when compared against each other. In the presentdisclosure, the assignment of amino acids to each domain is inaccordance with well-known conventions (e.g., Kabat, (1991) and/orChothia (1987)). The CDRs contain most of the residues which formspecific interactions with the antigen.

Tables 1 and 2 below depict the amino acid sequences and consensus aminoacid sequences encoding preferred CDRs for antibodies of the presentinvention.

TABLE 1 Fab LCDR1 LCDR2 LCDR3 JXB7 SASSSVSSTYLH RTSTLAS QQWSGYPFT (SEQID NO:26) (SEQ ID NO:30) (SEQ ID NO:31) 31B2 SASSSVSSTYLH RTSTLASQQWSGYPFT (SEQ ID NO:26) (SEQ ID NO:30) (SEQ ID NO:31) Hu22 SASSRVSSTYLFRTSTLAS QQWSGYPFT (SEQ ID NO:43) (SEQ ID NO:30) (SEQ ID NO:31) 1SLSSRVSSTYLF RTSTLAS QQWSGYPFT (SEQ ID NO:47) (SEQ ID NO:30) (SEQ IDNO:31) 2 SISSRVSSTYLF RTSTLAS QQWSGYPFT (SEQ ID NO:48) (SEQ ID NO:30)(SEQ ID NO:31) 3 SWSSRVSSTYLF RTSTLAS QQWSGYPFT (SEQ ID NO:49) (SEQ IDNO:30) (SEQ ID NO:31) 4 SAGSRVSSTYLF RTSTLAS QQWSGYPFT (SEQ ID NO:50)(SEQ ID NO:30) (SEQ ID NO:31) 5 SASSRVVSTYLF RTSTLAS QQWSGYPFT (SEQ IDNO:51) (SEQ ID NO:30) (SEQ ID NO:31) 6 SASSRVSSTYLF RTSPLAS QQWSGYPFT(SEQ ID NO:43) (SEQ ID NO:53) (SEQ ID NO:31) 7 SASSRVSSTYLF RTSALASQQWSGYPFT (SEQ ID NO:43) (SEQ ID NO:54) (SEQ ID NO:31) 8 SASSRVSSTYLFRTSWLAS QQWSGYPFT (SEQ ID NO:43) (SEQ ID NO:55) (SEQ ID NO:31) 9SASSRVSSTYLF RTSTGAS QQWSGYPFT (SEQ ID NO:43) (SEQ ID NO:56) (SEQ IDNO:31) 10 SASSRVSSTYLF RTSTLTS QQWSGYPFT (SEQ ID NO:43) (SEQ ID NO:57)(SEQ ID NO:31) 11 SASSRVSSTYLF RTSTLVS QQWSGYPFT (SEQ ID NO:43) (SEQ IDNO:58) (SEQ ID NO:31) 12 SASSRVSSTYLF RTSTLLS QQWSGYPFT (SEQ ID NO:43)(SEQ ID NO:59) (SEQ ID NO:31) 13 SASSRVSSTYLF RTSTLAS QQWSGYPFV (SEQ IDNO:43) (SEQ ID NO:30) (SEQ ID NO:61) *Consensus SX₁X₂SX₃VSSTYLFRTSX₄X₅X₆S QQWSGYPFX₇ (SEQ ID NO:52) (SEQ ID NO:60) (SEQ ID NO:62) *X₁is A, L, I, or W; X₂ is S or G; X₃ is R or 5; X₄ is T, P, A, or W; X₅ isL or G; X₆ is A, T, V, or L; X₇ is T or V

TABLE 2 Fab HCDR1 HCDR2 HCDR3 JXB7 GYTFTIYPIE NFHPYNGDTNYNEKFKG GGTGSFDY(SEQ ID NO:27) (SEQ ID NO:28) (SEQ ID NO:46) 31B2 GYTFYIYPISNFHPYKGLTNYNEKFKG GGTGSFDY (SEQ ID NO:29) (SEQ ID NO:33) (SEQ ID NO:46)Hu22 GYTFTIYPIS NFHPYLGDTNYNEKFKG GGTGSFDY (SEQ ID NO:32) (SEQ ID NO:44)(SEQ ID NO:46) 14 GYTFLIYPIS NFHPYLGDTNYNEKFKG GGTGSFDY (SEQ ID NO:63)(SEQ ID NO:44) (SEQ ID NO:46) 15 GYTFWIYPIS NFHPYLGDTNYNEKFKG GGTGSFDY(SEQ ID NO:64) (SEQ ID NO:44) (SEQ ID NO:46) 16 GYTFTIYPISNFHPYLGTTNYNEKFKG GGTGSFDY (SEQ ID NO:32) (SEQ ID NO:66) (SEQ ID NO:46)17 GYTFTIYPIS NFHPYLGLTNYNEKFKG GGTGSFDY (SEQ ID NO:32) (SEQ ID NO:67)(SEQ ID NO:46) 18 GYTFTIYPIS NFHPYLGVTNYNEKFKG GGTGSFDY (SEQ ID NO:32)(SEQ ID NO:68) (SEQ ID NO:46) 19 GYTFTIYPIS NFHPYLGMTNYNEKFKG GGTGSFDY(SEQ ID NO:32) (SEQ ID NO:69) (SEQ ID NO:46) 20 GYTFTIYPISNFHPYLGDANYNEKFKG GGTGSFDY (SEQ ID NO:32) (SEQ ID NO:70) (SEQ ID NO:46)21 GYTFTIYPIS NFHPYLGDTNYNEKFKG GGFGSFDY (SEQ ID NO:32) (SEQ ID NO:44)(SEQ ID NO:72) 22 GYTFTIYPIS NFHPYLGDTNYNEKFKG GGTGAFDY (SEQ ID NO:32)(SEQ ID NO:44) (SEQ ID NO:73) 23 GYTFTIYPIS NFHPYLGDTNYNEKFKG GGTGSFPY(SEQ ID NO:32) (SEQ ID NO:44) (SEQ ID NO:74) *Consensus GYTFX₈IYPI X₉NFHPYLGX₁₀X₁₁NYNEKFKG GGX₁₂GX₁₃FX₁₄Y (SEQ ID NO:65) (SEQ ID NO:71) (SEQID NO:75) *X₈ is T, W, Y, or L; X₉ is S or E; X₁₀ is D, T, L, V, or M;X₁₁ is T or A; X₁₂ is T or F; X₁₃ is S or A; X₁₄ is D or P

The SEQ ID NOs of the amino acid sequences and consensus amino acidsequences encoding more preferred CDRs for antibodies of the presentinvention are provided in Table 3 below.

TABLE 3 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID Fab NO: NO: NO: NO: NO: NO: 1.5 43 57 61 63 80 46 1.7 4358 61 32 81 46 1.10 43 53 61 63 82 46 1.13 43 57 61 63 83 46 1.15 43 3061 63 82 46 3.2 43 53 61 63 84 46 3.6 43 53 61 32 84 46 3.7 43 57 61 6385 46 3.8 43 53 31 63 84 46 3.9 43 53 61 63 86 46 3.12 43 57 61 63 84 463.23 43 53 61 63 85 46 Hu22 43 30 31 32 44 46 L1.5 41 53 31 63 84 46H1.39 43 53 31 78 84 46 *Consensus 42 76 62 79 87 46

Human light chain constant regions are classified as kappa or lambda andcharacterized by a particular constant region as known in the art. Humanheavy chain constant regions are classified as gamma, mu, alpha, delta,or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, andIgE, respectively and several of these may be further divided intosubclasses e.g., IgG₁, IgG₂, IgG₃, IgG₄. Each heavy chain type has aparticular constant region with a sequence readily known in the art.Light chain constant region kappa and heavy chain constant regions IgG₁,IgG₂, and IgG₄ are preferred constant regions in the antibodies of theinvention. Preferred human heavy chain constant regions for theantibodies of the present invention are the heavy chain constant regionamino acid sequences as shown in SEQ ID NOs: 90-94 and any variantthereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about15 amino acid changes (including substitutions, insertions ordeletions). More preferably, human heavy chain constant regions of theantibodies of the present invention are the heavy chain constant regionamino acid sequences as shown in SEQ ID NOs: 93 and 94. Most preferably,the human heavy chain constant region of the antibodies of the presentinvention is the heavy chain constant region amino acid sequence shownin SEQ ID NO: 93. Preferred human light chain constant regions of theantibodies of the present invention are the light chain constant regionkappa amino acid sequence shown in SEQ ID NO: 89 and any variant thereofhaving 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 amino acid changes(including substitutions, insertions or deletions). Most preferably, thehuman light chain constant region of the antibodies of the presentinvention is the light chain constant region kappa amino acid sequenceas shown in SEQ ID NO: 89.

As used herein, the “antigen-binding region” or “antigen-bindingportion” refers to that portion of an antibody molecule, within thevariable region, which contains the amino acid residues that interactwith an antigen and confer on the antibody its specificity and affinityfor the antigen. This antibody portion includes the framework amino acidresidues necessary to maintain the proper conformation of theantigen-binding residues.

The present invention includes an antibody that selectively bindshepcidin-25 with a K_(D) of about 800 pM or less as determined by SPR at25° C. and wherein the antibody comprises at least one CDR selected fromthe group consisting of i) a HCDR3 having the amino acid sequence asshown in SEQ ID NO: 75, and ii) a LCDR3 having the amino acid sequenceas shown in SEQ ID NO: 62. Preferably, such an antibody comprises sixCDRs comprising amino acid and/or consensus amino acid sequencesselected from the group consisting of: (i) LCDR1, LCDR2, LCDR3, HCDR1,HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs:52, 60, 62, 65, 71, and 75, respectively; and (ii) LCDR1, LCDR2, LCDR3,HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQID NOs: 42, 76, 62, 79, 87, and 46, respectively. More preferably, anantibody of the present invention comprises six CDRs selected from thegroup consisting of: (i) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3having the amino acid sequences as shown in SEQ ID NOs: 41, 53, 31, 63,84, and 46, respectively; (ii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, andHCDR3 having the amino acid sequences as shown in SEQ ID NOs: 43, 30,31, 32, 44, and 46, respectively; (iii) LCDR1, LCDR2, LCDR3, HCDR1,HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs:43, 53, 61, 63, 85, and 46, respectively; and (iv) LCDR1, LCDR2, LCDR3,HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQID NOs: 43, 57, 61, 63, 84, and 46, respectively. Even more preferably,the antibody of the invention comprises two heavy chain polypeptides andtwo light chain polypeptides, and wherein each of the heavy chainpolypeptides have the amino acid sequence as shown in SEQ ID NO: 9 andeach of the light chain polypeptides have the amino acid sequence asshown in SEQ ID NO: 17. Even more preferably, the antibody has an IC₅₀between about 100 nM and about 50 nM in the hepcidin-induced ferroportininternalization assay described in Example 3, a k_(off) rate from humanhepcidin-25 between about between about 5.5×10⁻⁴ s⁻¹ and about 2.0×10⁻⁴s⁻¹, as determined by SPR at 25° C., and selectively binds humanhepcidin-25 with a K_(D) between about 100 pM and about 50 pM. Mostpreferably, the antibody of the invention comprises two heavy chainpolypeptides and two light chain polypeptides, and wherein each of theheavy chain polypeptides have the amino acid sequence as shown in SEQ IDNO: 8 and each of the light chain polypeptides have the amino acidsequence as shown in SEQ ID NO: 16, wherein the antibody has an IC₅₀between about 100 nM and about 50 nM in the hepcidin-induced ferroportininternalization assay described in Example 3, a k_(off) rate from humanhepcidin-25 between about between about 5.5×10⁻⁴ s⁻¹ and about 2.0×10⁻⁴s⁻¹, as determined by SPR at 25° C., and selectively binds humanhepcidin-25 with a K_(D) between about 100 pM and about 50 pM.

Other preferred antibodies of the invention comprise a LCVR having anamino acid sequence selected from the group consisting of SEQ ID NOs:124, 125, 126, and 127. More preferably, an antibody of the inventioncomprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 148, 128, 150, and 151. Even more preferably,an antibody of the invention comprises a LCVR of SEQ ID NO: 126 and aHCVR of SEQ ID NO: 148. Even more preferably, an antibody of theinvention comprises a LCVR of SEQ ID NO: 127 and a HCVR of SEQ ID NO:128. Even more preferably, an antibody of the invention comprises a LCVRof SEQ ID NO: 125 and a HCVR of SEQ ID NO: 151. A most preferredantibody of the invention comprises a LCVR of SEQ ID NO: 124 and a HCVRof SEQ ID NO: 150. Such LCVRs are preferably linked to a light chainconstant region of human origin or derived from a light chain constantregion of human origin, preferably a human kappa chain, and mostpreferably a kappa chain of SEQ ID NO: 89. Such HCVRs are preferablylinked to a heavy chain constant region of human origin or derived froma heavy chain constant region of human origin, preferably IgG₁, IgG₂, orIgG₄, more preferably, a heavy chain constant region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 90, 91,92, 93, and 94, and most preferably, a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO: 93. Preferably, theantibody has an IC₅₀ between about 100 nM and about 50 nM in thehepcidin-induced ferroportin internalization assay described in Example3, a k_(off) rate from human hepcidin-25 between about between about5.5×10⁻⁴ s⁻¹ and about 2.0×10⁻⁴ s⁻¹ as determined by SPR at 25° C., andselectively binds human hepcidin-25 with a K_(D) between about 100 pMand about 50 pM.

An antibody of the invention may comprise a heavy chain polypeptidehaving an amino acid sequence as shown in SEQ ID NO: 6 and a light chainpolypeptide having an amino acid sequence as shown in SEQ ID NO: 14. Aheavy chain polypeptide having an amino acid sequence as shown in SEQ IDNO: 6 may be encoded by a polynucleotide sequence of SEQ ID NO: 2. Alight chain polypeptide having an amino acid sequence as shown in SEQ IDNO: 14 may be encoded by a polynucleotide having the nucleic acidsequence as shown in SEQ ID NO: 10.

An antibody of the invention comprises a heavy chain polypeptide havingan amino acid sequence as shown in SEQ ID NO: 7 and a light chainpolypeptide having an amino acid sequence as shown in SEQ ID NO: 15. Aheavy chain polypeptide having an amino acid sequence as shown in SEQ IDNO: 7 may be encoded by a polynucleotide sequence of SEQ ID NO: 3. Alight chain polypeptide having an amino acid sequence as shown in SEQ IDNO: 15 may be encoded by a polynucleotide having the nucleic acidsequence as shown in SEQ ID NO: 11.

An antibody of the invention also comprises a heavy chain polypeptidehaving an amino acid sequence as shown in SEQ ID NO: 8 and a light chainpolypeptide having an amino acid sequence as shown in SEQ ID NO: 16. Aheavy chain polypeptide having an amino acid sequence as shown in SEQ IDNO: 8 may be encoded by a polynucleotide sequence as shown in SEQ ID NO:4. A light chain polypeptide having an amino acid sequence as shown inSEQ ID NO: 16 may be encoded by a polynucleotide having the nucleic acidsequence as shown in SEQ ID NO: 12.

In another embodiment, an antibody of the invention comprises a heavychain polypeptide having an amino acid sequence as shown in SEQ ID NO: 9and a light chain polypeptide having an amino acid sequence as shown inSEQ ID NO: 17. A heavy chain polypeptide having an amino acid sequenceas shown in SEQ ID NO: 9 may be encoded by a polynucleotide sequence ofSEQ ID NO: 5. A light chain polypeptide having an amino acid sequence asshown in SEQ ID NO: 17 may be encoded by a polynucleotide having thenucleic acid sequence as shown in SEQ ID NO: 13.

Preferred human engineered antibodies of the invention are referred toherein as Mabs L1.5, Hu22, 3.12, and 3.23. The SEQ ID NOs of the aminoacid sequences encoding the heavy chains, the light chains, the heavyand light chain variable regions, and the CDRs for Mabs L1.5, Hu22,3.12, and 3.23 are provided in Table 4 below.

TABLE 4 Heavy Light HC HC HC LCV LC LC LC Mab Chain Chain HCVR CDR1 CDR2CDR3 R CDR1 CDR2 CDR3 L1.5 6 14 148 63 84 46 126 41 53 31 Hu22 7 15 12832 44 46 127 43 30 31 3.12 8 16 150 63 84 46 124 43 57 61 3.23 9 17 15163 85 46 125 43 53 61

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, isolated host cell lines producing an antibody of theinvention, culture these host cells and recover the antibody from theculture medium.

The present invention is also directed to host cells that express ananti-hepcidin antibody of the invention. A wide variety of hostexpression systems known in the art can be used to express an antibodyof the present invention including prokaryotic (bacterial) andeukaryotic expression systems (such as yeast, baculovirus, plant,mammalian and other animal cells, transgenic animals, and hybridomacells), as well as phage display expression systems.

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transformed, transduced,infected or the like with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and/or heavychains of the antibody such that the light and/or heavy chains areexpressed in the host cell. The heavy chain and the light chain may beexpressed independently from different promoters to which they areoperably-linked in one vector or, alternatively, the heavy chain and thelight chain may be expressed independently from different promoters towhich they are operably-linked in two vectors one expressing the heavychain and one expressing the light chain. Optionally, the heavy chainand light chain may be expressed in different host cells.

Additionally, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody light and/or heavychain from a host cell. The antibody light and/or heavy chain gene canbe cloned into the vector such that the signal peptide isoperably-linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide. Preferably, the recombinant antibodies aresecreted into the medium in which the host cells are cultured, fromwhich the antibodies can be recovered or purified.

An isolated DNA encoding a HCVR can be converted to a full-length heavychain gene by operably-linking the HCVR-encoding DNA to another DNAmolecule encoding heavy chain constant regions. The sequences of human,as well as other mammalian, heavy chain constant region genes are knownin the art. DNA fragments encompassing these regions can be obtainede.g., by standard PCR amplification. The heavy chain constant region canbe of any type, (e.g., IgG, IgA, IgE, IgM or IgD), class (e.g., IgG₁,IgG₂, IgG₃ and IgG₄) or subclass constant region and any allotypicvariant thereof as described in Kabat (supra).

An isolated DNA encoding a LCVR region may be converted to a full-lengthlight chain gene (as well as to a Fab light chain gene) by operablylinking the LCVR-encoding DNA to another DNA molecule encoding a lightchain constant region. The sequences of human, as well as othermammalian, light chain constant region genes are known in the art. DNAfragments encompassing these regions can be obtained by standard PCRamplification. The light chain constant region can be a kappa or lambdaconstant region.

In addition to the antibody heavy and/or light chain gene(s), arecombinant expression vector of the invention carries regulatorysequences that control the expression of the antibody chain gene(s) in ahost cell. The term “regulatory sequence” is intended to includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals), as needed, that control the transcription ortranslation of the antibody chain gene(s). The design of the expressionvector, including the selection of regulatory sequences may depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired. Preferred regulatory sequences formammalian host cell expression include viral elements that direct highlevels of protein expression in mammalian cells, such as promotersand/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40(SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP))and/or polyoma virus.

Additionally, the recombinant expression vectors of the invention maycarry additional sequences, such as sequences that regulate replicationof the vector in host cells (e.g., origins of replication) and one ormore selectable marker genes. The selectable marker gene facilitatesselection of host cells into which the vector has been introduced. Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin, or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (dhfr) gene (for use in dhfr-minushost cells with methotrexate selection/amplification), the neo gene (forG418 selection), and glutamine synthetase (GS) in a GS-negative cellline (such as NSO) for selection/amplification.

For expression of the light and/or heavy chains, the expressionvector(s) encoding the heavy and/or light chains is introduced into ahost cell by standard techniques e.g. electroporation, calcium phosphateprecipitation, DEAE-dextran transfection, transduction, infection andthe like. Although it is theoretically possible to express theantibodies of the invention in either prokaryotic or eukaryotic hostcells, eukaryotic cells are preferred, and most preferably mammalianhost cells, because such cells are more likely to assemble and secrete aproperly folded and immunologically active antibody. Preferred mammalianhost cells for expressing the recombinant antibodies of the inventioninclude Chinese Hamster Ovary (CHO cells) [including dhfr minus CHOcells, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216-20, 1980, used with a DHFR selectable marker, e.g. as describedin Kaufman and Sharp, J. Mol. Biol. 159:601-21, 1982], NSO myelomacells, COS cells, and SP2/0 cells. When recombinant expression vectorsencoding antibody genes are introduced into mammalian host cells, theantibodies are produced by culturing the host cells for a period of timesufficient to allow for expression of the antibody in the host cells or,more preferably, secretion of the antibody into the culture medium inwhich the host cells are grown under appropriate conditions known in theart. Antibodies can be recovered from the host cell and/or the culturemedium using standard purification methods.

Host cells can also be used to produce portions, or fragments, of intactantibodies, e.g., Fab fragments or scFv molecules by techniques that areconventional. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain of anantibody of this invention. Recombinant DNA technology may also be usedto remove some or all the DNA encoding either or both of the light andheavy chains that is not necessary for binding to human hepcidin-25. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention.

The invention provides a host cell comprising a nucleic acid molecule ofthe present invention. Preferably a host cell of the invention comprisesone or more vectors or constructs comprising a nucleic acid molecule ofthe present invention. For example, a host cell of the invention is acell into which a vector of the invention has been introduced, saidvector comprising a polynucleotide encoding a LCVR of an antibody of theinvention and/or a polynucleotide encoding a HCVR of the invention. Theinvention also provides a host cell into which two vectors of theinvention have been introduced; one comprising a polynucleotide encodinga LCVR of an antibody of the invention and one comprising apolynucleotide encoding a HCVR present in an antibody of the inventionand each operably-linked to enhancer/promoter regulatory elements (e.g.,derived from SV40, CMV, adenovirus and the like, such as a CMVenhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLPpromoter regulatory element) to drive high levels of transcription ofthe genes.

Once expressed, the intact antibodies, individual light and heavychains, or other immunoglobulin forms of the present invention can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, ion exchange, affinity (e.g., Protein A), reversephase, hydrophobic interaction column chromatography, hydroxylapatitechromatography, gel electrophoresis, and the like. Standard proceduresfor purification of therapeutic antibodies are described, for example,by Feng L1, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee in anarticle entitled “Current Therapeutic Antibody Production and ProcessOptimization” (BioProcessing Journal, September/October 2005), forexample. Additionally, standard techniques for removing viruses fromrecombinantly expressed antibody preparations are also known in the art(see, for example, Gerd Kern and Mani Krishnan, “Viral Removal byFiltration: Points to Consider” (Biopharm International, October 2006)).The effectiveness of filtration to remove viruses from preparations oftherapeutic antibodies is known to be at least in part dependent on theconcentration of protein and/or the antibody in the solution to befiltered. The purification process for antibodies of the presentinvention may include a step of filtering to remove viruses from themainstream of one or more chromatography operations. Preferably, priorto filtering through a pharmaceutical grade nanofilter to removeviruses, a chromatography mainstream containing an antibody of thepresent invention is diluted or concentrated to give total proteinand/or total antibody concentration of about 1 g/L to about 3 g/L. Evenmore preferably, the nanofilter is a DV20 nanofilter (e.g., PallCorporation; East Hills, N.Y.). Substantially pure immunoglobulins of atleast about 90%, about 92%, about 94% or about 96% homogeneity arepreferred, and about 98 to about 99% or more homogeneity most preferred,for pharmaceutical uses. Once purified, partially or to homogeneity asdesired, the sterile antibodies may then be used therapeutically, asdirected herein.

In view of the aforementioned discussion, the present invention isfurther directed to an antibody obtainable by a process comprising thesteps of culturing a host cell including, but not limited to amammalian, plant, bacterial, transgenic animal, or transgenic plant cellwhich has been transformed by a polynucleotide or a vector comprisingnucleic acid molecules encoding antibodies of the invention so that thenucleic acid is expressed and, optionally, recovering the antibody fromthe host cell culture medium. Preferably, the host cell comprises avector comprising a nucleic acid molecule encoding a light chainpolypeptide having the amino acid sequence as shown in SEQ ID NOs: 14,15, 16, or 17. More preferably, the host cell comprises a vectorcomprising a nucleic acid molecule as shown in SEQ ID NO: 12 or 13. Evenmore preferably, the transformed host cell is a Chinese hamster ovary,NSO myeloma, COS, or SP2/0 cell.

The present invention is further directed to a method of producing anantibody of the invention comprising the steps of transforming a hostcell including, but not limited to a mammalian, plant, bacterial,transgenic animal, or transgenic plant cell with a polynucleotide or avector comprising a nucleic acid molecule encoding an antibody of theinvention so that the nucleic acid is expressed and recovering theantibody from the host cell culture medium. Preferably, the host cell istransformed with a vector comprising a nucleic acid molecule encoding alight chain polypeptide having the amino acid sequence as shown in SEQID NOs: 14, 15, 16, or 17. More preferably, the host cell has beentransformed with a vector comprising a nucleic acid molecule as shown inSEQ ID NO: 12 or 13. Even more preferably, the host cell is a Chinesehamster ovary, NSO myeloma, COS, or SP2/0 cell.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the isolated polynucleotide (1)is not associated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, (2) is linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldnormally be found, (2) is essentially free of other proteins from thesame source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is not associated (bycovalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of synthetic origin, or any combination thereof.Preferably, the isolated protein is substantially purified from proteinsor polypeptides or other contaminants that are found in its naturalenvironment that would interfere with its use (therapeutic, diagnostic,prophylactic, research or otherwise).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, an antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.

As used herein, “substantially pure” or “substantially purified” means acompound or species that is the predominant species present (i.e., on amolar basis it is more abundant than any other individual species in thecomposition). In certain embodiments, a substantially purifiedcomposition is a composition wherein the species comprises at leastabout 50 percent (on a molar basis) of all macromolecular speciespresent. In certain embodiments, a substantially pure composition willcomprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolarspecies present in the composition. In certain embodiments, the speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The present invention further provides an isolated polynucleotide thatencodes the amino acid sequence selected from the group consisting ofSEQ ID NOs: 124, 125, 126, 127, 128, 148, 150, and 151.

In another embodiment, the present invention provides a recombinantexpression vector comprising polynucleotide that encodes the amino acidsequence selected from the group consisting of SEQ ID NOs: 124, 125,126, 127, 128, 148, 150, and 151.

The phrase “human engineered antibodies” as used herein refers to anantibody wherein at least one portion is of human origin. Furthermore,as used herein, the phrase “human engineered antibodies” refer to thespecific antibodies disclosed herein as well as additional antibodiesthat have similar functional properties according to the invention asthe antibodies disclosed herein and have framework regions that aresubstantially human or fully human surrounding CDRs that are derivedfrom a non-human antibody. Substantially human frameworks are those thathave at least 80% sequence identity to a known human germline frameworksequence. Preferably, the substantially human frameworks have at leastabout 85%, about 90%, about 95%, or about 99% sequence identity to aknown human germline framework sequence. Most preferably, humanengineered antibodies of the present invention contain minimal sequencederived from a non-human antibody.

For example, the human engineered antibody can comprise portions derivedfrom an antibody of nonhuman origin, such as a mouse, and portionsderived from an antibody of human origin, joined together, e.g.,chemically by conventional techniques (e.g., synthetic) or prepared as acontiguous polypeptide using genetic engineering techniques. As usedherein, the term “framework” when used in reference to an antibodyvariable region is entered to mean all amino acid residues outside theCDRs within the variable region of an antibody. As used herein, the term“framework region” is intended to mean each domain of the framework thatis separated by the CDRs. The framework regions for the light chain aresimilarly separated by each of the light chain variable region CDRs.Preferably, the light chain variable region and/or heavy chain variableregion comprises a framework or at least a portion of a framework region(e.g., containing 2 or 3 subregions, such as FR2 and FR3). Morepreferably, at least FRL1, FRL2, FRL3, or FRL4 is fully human or atleast FRH1, FRH2, FRH3, or FRH4 is fully human. Even more preferably, atleast FRL1, FRL2, FRL3, or FRL4 is a germline sequence (e.g., humangermline) or comprises human consensus sequences for the particularframework known in the art and/or at least FRH1, FRH2, FRH3, or FRH4 isa germline sequence (e.g., human germline) or comprises human consensussequences for the particular framework. In preferred embodiments, anantibody of the present invention comprises human germline light chainframework sequences and human germline heavy chain framework sequences(see, e.g., PCT WO 2005/005604). More preferably, human germline lightchain frameworks are selected from the group consisting of: A11, A17,A18, A19, A20, A27, A30, L1, L11, L12, L2, L5, L6, L8, O12, O18, O2, andO8. Even more preferably, the human germline light chain framework is O2or O8. Most preferably, the human germline light chain framework is O2.Additionally, preferred human germline heavy chain frameworks areselected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20,VH3-72, VH1-24, VH1-46, VH3-9, VH3-66, VH3-74, VH4-31, VH1-18, VH1-69,VH3-7, VH3-11, VH3-15, VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59,VH5-51, VH3-73, VH1-58, VH1-3, and VH1-2. Even more preferably, thehuman germline heavy chain framework is VH1-69 or VH1-18. Mostpreferably, the human germline heavy chain framework is VH1-69

Human engineered antibodies in addition to those disclosed herein thatselectively bind human hepcidin-25 with the functional propertiesaccording to the invention can be generated using several differentapproaches. The specific antibodies disclosed herein can be used as atemplate or parent antibody to make additional antibodies. In oneapproach the parent antibody CDRs are grafted into a human frameworkthat has a high sequence identity with the parent antibody framework.The sequence identity of the new framework will generally be at least80%, at least 85%, or at least 90% with the corresponding framework inthe parent antibody. This grafting may result in a reduction in bindingaffinity compared to the parent antibody. If this is the case, theframework can be back-mutated to the parent framework at certainpositions based on specific criteria published by Queen et al. Theidentification of residues to consider for back-mutation may be carriedout as follows: When an amino acid falls under the following category,the framework amino acid of the human germ-line sequence that is beingused (acceptor framework) is replaced by a framework amino acid from aframework of the parent antibody (donor framework):

(a) the amino acid in the human framework of the acceptor framework isunusual for human frameworks at that position, whereas the correspondingamino acid in the donor immunoglobulin is typical for human frameworksat that position;

(b) the position of the amino acid is immediately adjacent to one of theCDRs; or

(c) any side chain atom of a framework amino acid is within about 5-6angstroms (center-to-center) of any atom of a CDR amino acid in a threedimensional immunoglobulin model [Queen, et al., Proc. Natl. Acad. Sci.USA 88, 2869 (1991)].

When each of the amino acids in the human framework of the acceptorframework and a corresponding amino acid in the donor framework isunusual generally for human frameworks at that position, such an aminoacid may be replaced by an amino acid typical for human frameworks atthat position. This back-mutation criteria enables one to recover theactivity of the parent.

Another approach would be to randomly mutate the grafted CDRs withoutchanging the framework and screen such antibodies for binding affinitythat is as good or better than the parent antibody. Further, acombination of both these approaches is possible. After grafting,specific framework regions may be back-mutated in addition to makingchanges in the CDRs. This general methodology is described by Wu, etal., (1999), “Humanization of a murine monoclonal antibody bysimultaneous optimization of framework and CDR residues”, J. Mol. Biol.,294:151-162.

As used herein, the term “donor” is intended to mean a parent antibodymolecule or fragment thereof from which a portion is derived from, givenor contributes to another antibody molecule or fragment thereof so as toconfer either a structural or functional characteristic of the parentmolecule onto the receiving molecule. For the specific example of CDRgrafting, the parent molecule from which the grafted CDRs are derived isa donor molecule. The donor CDRs confer binding affinity of the parentmolecule onto the receiving molecule. It should be understood that adonor molecule does not have to be from a different species as thereceiving molecule of fragment thereof. Instead, it is sufficient thatthe donor is a separate and distinct molecule.

As used herein, the term “acceptor” is intended to mean an antibodymolecule or fragment thereof which is to receive the donated portionfrom the parent or donor antibody molecule or fragment thereof. Anacceptor antibody molecule or fragment thereof is therefore impartedwith the structural or functional characteristic of the donated portionof the parent molecule. For the specific example of CDR grafting, thereceiving molecule for which the CDRs are grafted is an acceptormolecule. The acceptor antibody molecule or fragment is imparted withthe binding affinity of the donor CDRs or parent molecule. As with adonor molecule, it is understood that an acceptor molecule does not haveto be from a different species as the donor.

A “variable region” when used in reference to an antibody or a heavy orlight chain thereof is intended to mean the amino terminal portion of anantibody which confers antigen binding onto the molecule and which isnot the constant region. The term is intended to include functionalfragments thereof which maintain some of all of the binding function ofthe whole variable region. Therefore, the term “heteromeric variableregion binding fragments” is intended to mean at least one heavy chainvariable region and at least one light chain variable regions orfunctional fragments thereof assembled into a heteromeric complex.Heteromeric variable region binding fragments include, for example,functional fragments such as Fab, F(ab)₂, Fv, single chain Fv (scFv) andthe like. Such functional fragments are well known to those skilled inthe art. Accordingly, the use of these terms in describing functionalfragments of a heteromeric variable region is intended to correspond tothe definitions well known to those skilled in the art. Such terms aredescribed in, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biologyand Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.),New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics,22:189-224 (1993); Pl{umlaut over (υ)}ckthun and Skerra, Meth. Enzymol.,178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, SecondEd., Wiley-Liss, Inc., New York, N.Y. (1990).

Preferably, a human engineered antibody has CDRs that originate from orare derived from a parent antibody, i.e., a non-human antibody,preferably a mouse antibody or fragment thereof such as the mouse FabJXB7, while framework and constant region, to the extent it is present,(or a significant or substantial portion thereof, i.e., at least about90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) are encoded by nucleic acidsequence information that occurs in the human germline immunoglobulinregion (see, e.g., the International ImMunoGeneTics Database) or inrecombined or mutated forms thereof whether or not said antibodies areproduced in a human cell. Preferably, at least two, three, four, five orsix CDRs of a human engineered antibody are optimized from the CDRs of anon-human parent antibody from which the human engineered antibody wasderived, to generate a desired property, e.g., improved specificity,affinity or neutralization, which may be identified by a screeningassay, e.g., an ELISA assay. Preferably, an antibody of the inventioncomprises a HCDR3 identical to the HCDR3 of the parent mouse Fab JXB7(i.e., SEQ ID NO: 46) and HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 compriseat least one amino acid substitution when compared to that present inthe parent mouse Fab JXB7. Certain amino acid substitutions in the CDRsof human engineered antibodies of the invention as compared to those ofthe parent mouse Fab JXB7 decrease the likelihood of instability of theantibody (e.g., removal of one or more CDR Asn residues) or decrease thelikelihood of immunogenicity of the antibody when administered to ahuman subject (e.g., as predicted by IMMUNOFILTER™ Technology (Xencor,Inc., Monrovia, Calif.)).

Human engineered antibodies may be subjected to in vitro mutagenesisusing methods of routine use in the art and, thus, the framework regionamino acid sequences of the HCVRs and LCVRs of the human engineeredrecombinant antibodies are sequences that, while derived from thoserelated to human germline HCVR and LCVR sequences, may not naturallyexist within the human antibody germline repertoire in vivo. It iscontemplated that such amino acid sequences of the HCVR and LCVRframeworks of the human engineered recombinant antibodies are at leastabout 85%, about 90%, about 92%, about 94%, about 95%, about 96%, about98% or, more preferably, at least about 99% or, most preferably, 100%identical to a human germline sequence. Accordingly, human engineeredantibodies may comprise residues which are found neither in therecipient antibody nor in the CDR or framework sequences imported fromthe parent antibody.

There are multiple methods available in the art to generate humanengineered antibodies (see, e.g., PCT International Patent ApplicationPublication No. WO2006/06046935; Queen, et al., Proc. Natl. Acad. Sci.USA 88:2869 (1991); Jones et al., Nature, 321:522 (1986); Riechmann etal., Nature, 332:323-327 (1988); and Verhoeyen et al., Science, 239:1534(1988)). For example, human engineered antibodies may be produced byobtaining nucleic acid sequences encoding the HCVR and LCVR of a parentantibody (e.g., a murine antibody or antibody made by a hybridoma) whichselectively binds hepcidin-25, identifying the CDRs in said HCVR andLCVR (nonhuman), and grafting such CDR-encoding nucleic acid sequencesonto selected human framework-encoding nucleic acid sequences.Optionally, a CDR region may be optimized by mutagenizing randomly or atparticular locations in order to substitute one or more amino acids inthe CDR with a different amino acid prior to grafting the CDR regioninto the framework. Alternatively, a CDR region may be optimizedsubsequent to insertion into the human framework using methods availableto one of skill in the art.

After the CDR-encoding sequences are grafted onto the selected humanframework encoding sequences, the resultant DNA sequences encoding thehuman engineered variable heavy and variable light sequences are thenexpressed to produce a human engineered antibody that selectively bindshepcidin-25. The human engineered HCVR and LCVR may be expressed as partof a whole anti-hepcidin-25 antibody molecule, i.e., as a fusion proteinwith human constant domain sequences. However, the HCVR and LCVRsequences can also be expressed in the absence of constant sequences toproduce a human engineered anti-hepcidin-25 selective Fv or Fab, forexample (see, e.g., Watkins, J., et al., Anal. Biochem., 253:37-45(1997) and Watkins, J., et al., Anal. Biochem. 256:169-177, (1998)).

It will be appreciated that applying the teaching of the presentinvention the person skilled in the art may use common techniques e.g.site directed mutagenesis, to substitute, add, or deleteamino acidswithin the specific CDR and framework sequences herein disclosed and inso doing generate further variable region amino acid sequences derivedfrom the sequences herein provided. Up to all 21 alternative naturallyoccurring amino acids may be introduced at a specific substitution site.Finally, in vitro or in vivo screening technologies such as thosedescribed in Example 2 herein are available to the artisan for theselection of variable region amino acid sequences for Fab fragmentshaving the desired binding affinity to hepcidin polypeptides. In thisway further Fab fragments may be identified that are suitable forpreparing an anti-hepcidin antibody in accordance with the presentinvention. Preferably, amino acid substitution, addition, and deletionwithin the frameworks is restricted to one, two, three, or fourpositions within one or each of the framework region sequences (i.e.,FRL1, FRL2, FRL3, FRL4, FRH1, FRH2, FRH3, FRH4) disclosed herein.Preferably, amino acid substitution, addition, and deletion within theCDRs is restricted to one to three positions within one or each CDR,more preferably substitution, addition, and deletion at one or two aminoacid positions within one or each CDR is performed. Further preferred,amino acid substitution, addition, and deletion is performed at one ortwo amino acid positions in the CDRs of the heavy chain variable region.Most preferably amino acid substitution, addition, and deletion isperformed at one or two amino acid positions within CDRH2.

The resultant DNA sequences encoding the human engineered variable heavyand variable light sequences are then expressed to produce a humanengineered antibody that selectively binds human hepcidin-25 with highaffinity. The human engineered HCVR and LCVR may be expressed as part ofa whole anti-hepcidin-25 antibody molecule, i.e., as a fusion proteinwith human constant domain sequences.

Another aspect of the invention provides methods of using the antibodiesof the invention in relatively simple yet highly sensitive and selectiveimmunoassays for the detection and measurement of mature hepcidin inhuman tissues and biological fluids for diagnostic and prognosticpurposes.

The antibodies of the present invention provide the means to accuratelydetect or determine the amounts of mature hepcidin in a tissue orbiological fluid from a human for assessment of predispositions tomature hepcidin-promoteddisorders, and for detection and diagnosis ofsuch disorders in patients suffering therefrom. For example, theantibodies of the invention can be incorporated into sensitive andreliable immunoassays such as ELISA, RIA, immunodiffusion assays, orimmuno-detection assays, such as SPR assays. Similarly, the antibodiesof the present invention are also useful for immunohistochemical (IHC)and immunofluorescence (IF) assays of tissue or biological fluidsamples. Such analyses can be used to detect aberrant levels ofhepcidin-25 and hence to diagnose hepcidin-25 promoted disorders. Morespecifically, the present invention provides methods of diagnosing ahuman mature hepcidin-promoted disorder in a patient by determining thelevel of human mature hepcidin in a sample of tissue or a biologicalfluid from the patient and comparing the level of human mature hepcidinin the sample with the level of human mature hepcidin in a correspondingsample from one or more control individuals or with a reference standardthereby detecting a disorder associated with elevated levels of humanmature hepcidin. The disease state may comprise one or more of a geneticor non-genetic disease associated with decreased serum iron levels,reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit.Preferably the disease state may comprise one or more of a disorderassociated with anemia.

A method of monitoring a human mature hepcidin-promoted disease,disorder or condition in a patient is also provided. The method includesdetermining the level of human mature hepcidin in a sample of a tissueor biological fluid from a patient suffering from, or at risk of, ahuman mature hepcidin-promoted disease, disorder or condition at a firsttime point; determining the level of human mature hepcidin in one ormore samples of tissue or biological fluid from the patient at one ormore different time points; comparing the levels of human maturehepcidin determined at different time points and thereby monitoring thehuman mature hepcidin-promoted disease or condition.

The human mature hepcidin selective antibodies of the present inventionare particularly useful when applied to high-throughput methods. Suchmethods include micro-chip and micro-array methods, such that manysamples can be tested on a microplate or slide, or other assay substrateknown in the art.

The presence of human mature hepcidin or levels thereof in a biologicalsample may be established by combining the biological sample with, e.g.an antibody of the invention under conditions suitable to form anantigen-antibody complex. The antibody is directly or, more preferably,indirectly labeled with a detectable moiety to facilitate detection ofthe bound or unbound antibody. A wide variety of methods of detection ofimmunocomplex formation are well known in the art, for example, ELISA,RIA, immunoblot (e.g., dot blot, slot blot, western blot, etc.),indirect immunofluorescence techniques and methods that rely ondetection of changes in physical parameters, such as for instance, SPR,and the like. Such applications include methods that utilize ahepcidin-25 selective antibody of the invention conjugated with adetectable moiety to detect hepcidin in a biological sample, e.g., in ahuman biological fluid or in a cell or tissue extract. Antibodies of theinvention may be used in such assays with or without modification with adetectable moiety. If modified with a detectable moiety, antibodies ofthe invention may be modified by covalent or non-covalent attachment ofthe detectable moiety. As used herein, the term “detectable” describes afeature of a substance (a conjugate, compound, or moiety) that allowsidentifying or tracing the substance by a detector, using knownanalytical techniques. Representative examples of detectable moietiesinclude, without limitation, chromophores, fluorescent moieties,phosphorescent moieties, luminescent moieties, radioactive moieties,various enzymes (such as alkaline phosphatase, or horseradishperoxidase), magnetic moieties (e.g., diamagnetic, paramagnetic andferromagnetic materials), and heavy metal clusters, as well as any otherknown detectable moieties. The amount of an antibody-antigen standardcomplex formed may be quantitated by various methods known in the art,such as, e.g. photometric or calorimetric means. Preferably, theantibodies of the invention are used without modification, i.e.,indirectly labeled, according to methods well known in the art.

The invention embodies a method for detecting human mature hepcidinprotein in a biological sample, comprising incubating an antibody of theinvention with a biological sample under conditions and for a timesufficient to permit said antibody to bind to human mature hepcidinproteins, and detecting said binding.

The present invention also provides compositions, methods and kits forscreening samples suspected of containing human mature hepcidinpolypeptides. Such screening may be performed on patient samples, orlaboratory samples suspected of containing or producing suchpolypeptide. A kit can contain a hepcidin-25 selective antibody of thepresent invention. The kit can contain a suitable buffer and reagentsfor detecting an interaction between a sample and a hepcidin-25selective antibody of the present invention. The provided reagent can beradiolabeled, fluorescently-labeled or enzymatically-labeled agentcapable of binding or interacting with an antibody of the presentinvention such as an anti-mouse IgG antibody.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. When the reagent is provided in aliquid solution, preferably, the liquid solution is an aqueous solution.Preferably, when the reagent provided is attached to a solid support,the solid support can be chromatographic media, a test plate having aplurality of wells, or a microscope slide. When the reagent provided isa dry powder, the powder can be reconstituted by the addition of asuitable solvent, which may be provided in the kit as well.

The kit of the invention is provided in a container that generallyincludes a vial into which the antibody, antigen or detection reagentmay be placed, and preferably suitably aliquotted. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and reagent containers for commercial sale. Suchcontainers may include plastic containers into which the desired vialsare retained and one or more necessary chemicals, such as chromatographymaterial, solvents and eluents, test tubes, detergents, antibodies andchemicals for the detection reaction.

An antibody of the invention may be used to diagnose a disorder ordisease associated with the activity of mature hepcidin. In a similarmanner, the antibody of the invention can be used in an assay to monitorlevels of mature hepcidin in a subject being treated for a condition,disease, or disorder promoted mature hepcidin. Such applications includemethods that utilize an antibody of the invention and a label to detectmature hepcidin in a biological sample, e.g., in a human body fluid orin a cell or tissue extract. Antibodies of the invention may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a detectable moiety.

A variety of conventional protocols for measuring protein levels in abiological sample, including e.g. ELISAs, RIAs, and FACS, are known inthe art and provide a basis for diagnosing altered or abnormal levels ofmature hepcidin expression. Normal or standard hepcidin levels presentin a sample are established using any known technique, e.g., bycombining a sample comprising a mature hepcidin polypeptide with, e.g.an antibody of the invention under conditions suitable to form anantigen:antibody complex. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials. The amount of a standard complex formed isquantitated by various methods, such as, e.g., photometric means.Amounts of mature hepcidin polypeptide present in samples are thencompared with the standard values. A preferred antibody for use indiagnostic, prognostic, and/or monitoring assays, kits, and methods has(i) a heavy chain polypeptide having an amino acid sequence as shown inSEQ ID NO: NO: 6 and a light chain polypeptide having an amino acidsequence as shown in SEQ ID NO: 14; (ii) a heavy chain polypeptidehaving an amino acid sequence as shown in SEQ ID NO: 7 and a light chainpolypeptide having an amino acid sequence as shown in SEQ ID NO: 15;(iii) a heavy chain polypeptide having an amino acid sequence as shownin SEQ ID NO: 9 and a light chain polypeptide has the amino acidsequence as shown in SEQ ID NO: 17; or (iv) a heavy chain polypeptidehaving an amino acid sequence as shown in SEQ ID NO: 8 and a light chainpolypeptide has the amino acid sequence as shown in SEQ ID NO: 16.

An isolated hepcidin-25 selective antibody of the invention may be usedin therapy, preferably, human therapy.

A pharmaceutical composition comprising an antibody of the invention maybe used to increase serum iron levels, reticulocyte count, red bloodcell count, hemoglobin, and/or hematocrit in a human when an effectiveamount is administered to a human subject in need thereof. Furthermore,an antibody of the invention may be useful for the treatment ofconditions, diseases, or disorders wherein the presence of hepcidin-25causes or contributes to undesirable pathological effects or a decreaseof hepcidin-25 levels or hepcidin-25 bioactivity has a therapeuticbenefit in human subjects. Such conditions, diseases or disordersinclude, but are not limited to, anemia including, but not limited to,anemia resulting from infection, inflammation, chronic disease, and/orcancer. Subjects may be male or female.

The present invention includes a method of increasing serum iron levels,reticulocyte count, red blood cell count, hemoglobin, and/or hematocritthat comprises administering to a human subject in need thereof, aneffective amount of an antibody of the present invention thatselectively binds human hepcidin-25 with a K_(D) of about 800 pM orless. Additionally, or alternatively, the present invention includes amethod for treating a disease, condition or disorder, in a humansubject, which benefits from an increase in serum iron levels,reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit,including, but not limited to, anemia, e.g., anemia resulting frominfection, inflammation, chronic disease, and/or cancer. Preferably, thesubject has or is at risk of having undesirably low serum iron level,low reticulocyte count, red blood cell count, hemoglobin, and/orhematocrit. More preferably, the subject is at risk for, or sufferingfrom, anemia including, but not limited to, anemia resulting frominfection, inflammation, chronic disease, and/or cancer. Even morepreferably, the antibody comprises a LCVR comprising;

i) a LCDR1 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 41 and 43;

ii) a LCDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 30, 53, 56, 57, 58, and 76; and

iii) a LCDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 31, 61, and 60; and a HCVR comprising;

i) a HCDR1 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 32, 63, 78, and 79;

ii) a HCDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 80, 81, 82, 83, 84, 85, 86, and 87; and

iii) a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 46.Even more preferably, the antibody comprises a heavy chain and a lightchain polypeptide having (i) the amino acid sequences as shown in SEQ IDNOs: 6 and 14, respectively; (ii) the amino acid sequences as shown inSEQ ID NOs: 7 and 15, respectively; (iii) the amino acid sequences asshown in SEQ ID NOs: 9 and 17, respectively; or (iv) amino acidsequences as shown in SEQ ID NOs: 8 and 16, respectively. Mostpreferably, the antibody comprises a heavy chain and a light chainpolypeptide having the amino acid sequences as shown in SEQ ID NOs: 8and 16, respectively.

Additionally, the use of an antibody of the invention for thepreparation of a medicament for the treatment of anemia or at least oneof the aforementioned disorders is contemplated. Preferably, theantibody comprises a LCVR comprising;

i) a LCDR1 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 41 and 43;

ii) a LCDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 30, 53, 56, 57, 58, and 76; and

iii) a LCDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 31, 61, and 60; and a HCVR comprising;

i) a HCDR1 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 32, 63, 78, and 79;

ii) a HCDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 80, 81, 82, 83, 84, 85, 86, and 87; and

iii) a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 46.More preferably, the antibody comprises a heavy chain and a light chainpolypeptide having (i) the amino acid sequences as shown in SEQ ID NOs:6 and 14, respectively; (ii) the amino acid sequences as shown in SEQ IDNOs: 7 and 15, respectively; (iii) the amino acid sequences as shown inSEQ ID NOs: 9 and 17, respectively; or (iv) amino acid sequences asshown in SEQ ID NOs: 8 and 16, respectively. Most preferably, theantibody comprises a heavy chain and a light chain polypeptide havingthe amino acid sequences as shown in SEQ ID NOs: 8 and 16, respectively.

The term “treating” (or “treatment” and “treat” are intended to refer toall processes wherein there may be a slowing, interrupting, arresting,controlling, or stopping of the progression of the disorders describedherein, but does not necessarily indicate a total elimination of alldisorder symptoms. “Treatment”, as used herein, includes administrationof a compound of the present invention for treatment of a disease orcondition in a mammal, particularly in a human, and includes: (a)inhibiting further progression of the disease, i.e., arresting itsdevelopment; and (b) relieving the disease, i.e., causing regression ofthe disease or disorder or alleviating symptoms or complicationsthereof. Dosage regimens may be adjusted to provide the optimum desiredresponse (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

The term “preventing” (or “prevent” or “prevention”) means prohibiting,restraining, or inhibiting the incidence or occurrence of a symptom,disorder, condition, or disease. Acute events and chronic conditions maybe treated and prevented. In an acute event, an antibody of theinvention is administered at the onset of a symptom, disorder,condition, or disease, and is discontinued when the acute event ends. Incontrast, a chronic symptom, disorder, condition, or disease is treatedover a more protracted time frame.

A “disorder” is any condition that would benefit from treatmentaccording to the present invention. The terms “disorder”, “condition”and “disease” are used interchangeably herein and include chronic andacute mature hepcidin-promoted disorders, including, but not limited to,anemia including, but not limited to, anemia of chronic disease,including, but not limited to, anemia resulting from infection,inflammation, and/or cancer.

An antibody of the invention can be incorporated into a pharmaceuticalcomposition suitable for administration to a human subject. An antibodyof the invention may be administered to a human subject alone or incombination with a pharmaceutically acceptable carrier and/or diluent insingle or multiple doses. Such pharmaceutical compositions are designedto be appropriate for the selected mode of administration, andpharmaceutically acceptable diluents, carrier, and/or excipients such asdispersing agents, buffers, surfactants, preservatives, solubilizingagents, isotonicity agents including but not limited to sodium chloride,stabilizing agents and the like are used as appropriate. Saidcompositions can be designed in accordance with conventional techniquesdisclosed in, e.g., Remington, The Science and Practice of Pharmacy,19^(th) Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1995which provides a compendium of formulation techniques as are generallyknown to practitioners. Suitable carriers for pharmaceuticalcompositions include any material which, when combined with an antibodyof the invention, retains the molecule's activity and is non-reactivewith the subject's immune system. In certain embodiments, apharmaceutical composition of the present invention comprises i) anantibody of the invention, ii) a citrate buffer, and iii) sodiumchloride. Preferably, the antibody is present at a concentration rangingfrom about 1 mg/ml to about 35 mg/ml, citrate is present at aconcentration ranging from about 5 mM to about 20 mM, sodium chloride ispresent at a concentration ranging from about 100 mM to about 300 mM,and the pH of the composition is between about 5.0 to about 7.2. Morepreferably, the antibody is present at a concentration ranging fromabout 5 mg/ml to about 30 mg/ml, citrate is present at a concentrationranging from about 5 mM to about 15 mM, sodium chloride is present at aconcentration ranging from about 150 mM to about 300 mM, and the pH ofthe composition is between about 5.5 to about 6.5. Even more preferably,the antibody is present at a concentration ranging from about 5 mg/ml toabout 25 mg/ml, citrate is present at about 10 mM, sodium chloride ispresent at a concentration ranging from about 200 m M to about 300 mM,and the pH of the composition is between about 5.5 to about 6.5.

A pharmaceutical composition comprising an anti-hepcidin-25 antibody ofthe present invention can be administered to a subject at risk for orexhibiting pathologies as described herein, e.g., anemia disorders,using standard administration techniques.

The phrase “effective amount” as used herein refers to an amountnecessary (at dosages and for periods of time and for the means ofadministration) to achieve the desired therapeutic result. An effectiveamount of the antibody may vary according to factors such as the diseasestate, age, gender, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. An effective amount is also one in which any toxic ordetrimental effect of the antibody, are outweighed by thetherapeutically beneficial effects.

An effective amount is at least the minimal amount, but less than atoxic amount, of an active agent which is necessary to imparttherapeutic benefit to a subject. Stated another way, an effectiveamount or therapeutically effective amount of an antibody of theinvention is an amount which in mammals, preferably humans, (i)increases serum iron levels, reticulocyte count, red blood cell count,hemoglobin, and/or hematocrit, or (ii) treats a disorder wherein thepresence of mature hepcidin causes or contributes to an undesirablepathological effect, or (iii) a decrease in mature hepcidin levels ormature hepcidin bioactivity results in a beneficial therapeutic effectin a mammal, preferably a human, including, but not limited to, anemiaincluding, but not limited to, anemia of chronic disease, including, butnot limited to, anemia resulting from infection, inflammation, and/orcancer. An effective amount of an antibody of the invention may beadministered in a single dose or in multiple doses. Furthermore, aneffective amount of an antibody of the invention may be administered inmultiple doses of amounts that would be less than an effective amount ifnot administered more than once.

As is well known in the medical arts, dosages for any one subjectdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, gender, time androute of administration, general health, and other drugs beingadministered concurrently. Dose may further vary depending on the typeand severity of the disease. A typical dose can be, for example, in therange of about 1 mg to about 200 mg; preferably, about 2 mg to about 200mg; more preferably, about 5 mg to about 200 mg; even more preferably,about 5 mg to about 50 mg, even more preferably, about 5 mg to about 25mg; even more preferably, about 5 mg to about 20 mg; even morepreferably, about 5 mg to about 15 mg; however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. A daily parenteral dosage regimen can be fromabout 10 μg/kg to about 20 mg/kg, preferably from about 25 μg/kg toabout 20 mg/kg, more preferably from about 50 μg/kg to about 20 mg/kg,even more preferably, from about 100 μg/kg to about 20 mg/kg, even morepreferably from about 200 μg/kg to about 20 mg/kg, even more preferably,from about 300 μg/kg to about 20 mg/kg, even more preferably, from about400 μg/kg to about 20 mg/kg, even more preferably from about 500 μg/kgto about 20 mg/kg, from about 600 μg/kg to about 20 mg/kg, from about700 μg/kg to about 20 mg/kg, from about 800 μg/kg to about 20 mg/kg,from about 900 μg/kg to about 20 mg/kg, even more preferably, from about1 mg/kg to about 20 mg/kg, even more preferably, from about 2 mg/kg toabout 20 mg/kg, even more preferably, from about 3 mg/kg to about 20mg/kg, even more preferably, from about 4 mg/kg to about 20 mg/kg, evenmore preferably, from about 5 mg/kg to about 20 mg/kg, even morepreferably, from about 6 mg/kg to about 20 mg/kg, even more preferably,from about 7 mg/kg to about 20 mg/kg, and even more preferably, fromabout 8 mg/kg to about 20 mg/kg. Progress may be monitored by periodicassessment, and the dose adjusted accordingly.

These suggested amounts of antibody are subject to a great deal oftherapeutic discretion. The key factor in selecting an appropriate doseand scheduling is the result obtained. Factors for consideration in thiscontext include the particular disorder being treated, the clinicalcondition of the individual patient, the cause of the disorder, the siteof delivery of the antibody, the particular type of antibody, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

The route of administration of an antibody of the present invention maybe oral, parenteral, by inhalation, or topical. Preferably, theantibodies of the invention can be incorporated into a pharmaceuticalcomposition suitable for parenteral administration. The term parenteralas used herein includes intravenous, intramuscular, subcutaneous,rectal, vaginal, or intraperitoneal administration. Parenteral deliveryby intravenous or intraperitoneal or subcutaneous injection ispreferred. Subcutaneous injection is most preferred. Suitable vehiclesfor such injections are well known in the art.

The pharmaceutical composition typically must be sterile and stableunder the conditions of manufacture and storage in the containerprovided, including e.g., a sealed vial, syringe or other deliverydevice, e.g., a pen. Therefore, pharmaceutical compositions may besterile filtered after making the formulation, or otherwise mademicrobiologically acceptable.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention.

EXAMPLES Example 1 Production of Human Hepcidin-25

Human hepcidin-25 can be obtained from commercial sources (e.g., PeptideInternational (Louisville, Ky.)) or produced by a variety of syntheticor recombinant techniques known in the art. Alternatively, a fusionprotein comprising the twenty-five amino acids of human hepcidin-25sequence and having the amino acid sequence as shown in SEQ ID NO: 95 isexpressed in E. coli. Inclusion bodies are isolated from 3 liters of E.coli expressing the human hepcidin fusion protein after a 3-6 hourinduction with 1 mM IPTG at 37° C. The inclusion bodies are solubilizedin buffer A (50 mM Tris and 8 M urea (pH 8.0)). The supernatant ispassed over an IMAC column (20 mL resin). The column is washed withbuffer A until the absorbance returned to baseline and the boundpolypeptides are batch eluted from the column by 0.5 M imidazole inbuffer A. The human hepcidin-25 fusion protein is pooled and reducedwith 50 mM DTT. This fusion protein is then refolded by diluting pooledmaterial into 2 M urea, 3 mM cysteine, 50 mM Tris (pH 8.0) to a finalprotein concentration less than 50 μg/mL. This material is stirred atroom temperature and air oxidized for 48 hours. The oxidizedpolypeptides are passed over an IMAC column (20 mL) at a flow rate of 5mL/min, and the human hepcidin-25 fusion protein is batch eluted fromthe column by 0.5 M imidazol in buffer A. The pooled fractionscontaining the human hepcidin-25 fusion protein are concentrated andpassed over a Superdex 75 (GE Healthcare, XK26/60) sizing columnequilibrated with 50 mM Tris, 4 M urea, pH 8.0, at a flow rate of 3mL/min. The monomeric fusion protein is pooled and then diluted to 50 mMTris, 2M urea, 5 mM CaCl₂, pH 8.0 and then is cleaved with enterokinaseto produce human hepcidin-25 of SEQ ID NO: 1. Uncleaved humanhepcidin-25 fusion protein is removed by passive IMAC chromatography (asoutlined above). The flow through from the IMAC column is then passedover a C-18 Reversed Phase column at a flow rate of 4.0 mL/minute. Thecolumn is washed with 0.1% TFA in water until the absorbance returned tobaseline and the bound polypeptides are eluted from the column with alinear gradient of ACN from 20% to 40% with 0.1% TFA at a rate of0.5%/min. Fractions which contain the human hepcidin-25 polypeptide arepooled and analyzed by N-terminal amino acid sequencing and matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS).Polypeptides encoding rat, mouse, and cynomolgous monkey hepcidin-25 andvarious N-terminally truncated forms of human hepcidin-25, includinghepcidin-22 and hepcidin-20 were obtained commercially (e.g., PeptideInternational).

Example 2 Affinity Binding Measurements of anti-Hepcidin-25 Fabs andMabs

A surface plasmon resonance biosensor such as the BIAcore® T100 may beused to measure binding kinetics and affinity of the antibodiesdisclosed herein. The BIAcore® system utilizes the optical properties ofSPR to detect alteration in protein concentration of interactingmolecules within a dextran biosensor matrix. Except as noted, allreagents and materials are purchased from BIAcore® AB (Upsala, Sweden).All measurements are performed at 25° C. Samples are dissolved in HBS-EPbuffer (150 mM sodium chloride, 3 mM EDTA, 0.05% (w/v) surfactant P-20,and 10 mM HEPES, pH 7.4). To capture Fabs with human kappa,goat-anti-human kappa is immobilized on flow cells 1 to 4 of a CM5sensor chip at a level of 5000-10000 response units (Rus) using an aminecoupling kit. To capture Mabs with mouse IgG1, goat-anti-mouse Fc gammais immobilized on flow cells 1 to 4 of a CM5 sensor chip at a level of5000-10000 Rus using an amine coupling kit. To capture antibodies withhuman IgG4, protein A is immobilized on flow cells 1 to 4 of a CM4sensor chip at a level of 400-700 Rus using an amine coupling kit. Fabsprepared from E. coli periplasma and Mabs prepared from mammalian cellculture are evaluated using multiple analytical cycles. Each cycleconsists of the following steps: 0.3-2 minutes injection of a Fab or aMab at ˜10 μL/minute aiming at a capture of 200-1000 Rus, 2 minutesinjection at 50 μL/minute of various concentrations of human hepcidin-25(from 600 nM to 0.1 nM) obtained as described in Example 1 abovefollowed by 2-10 minutes for dissociation, and regeneration using 30 μLof 10 mM glycine hydrochloride, pH 1.5. The measurements are obtained at25° C. and the association and dissociation rates for each cycle areevaluated using a “1:1 with mass transfer” binding model in theBLAevaluation software.

The human hepcidin-25 binding parameters of the mouse Fab JXB7 andcertain human engineered anti-hepcidin Fabs are shown in Table 5. Thehuman hepcidin-25 binding affinity (K_(D)) of the other human engineeredFabs listed in Table 3 was determined to be between about 214 μM andabout 54 μM with each having a k_(off) rate from human hepcidin-25between about 7.68×10⁻⁴ s⁻¹ and about 2.22×10⁻⁴ s⁻¹. Therefore, humanengineered anti-hepcidin Fabs having a K_(D) for human hepcidin-25 up to52-fold lower than that of mouse Fab JXB7 were identified. The humanengineered anti-hepcidin Fabs shown in Table 5 comprise the humangermline light and heavy chain frameworks O2 and VH1-69, respectively.

TABLE 5 Binding Properties to Human Hepcidin-25 Fab K_(on) (M⁻¹, s⁻¹)K_(off) (s⁻¹) K_(D) (M) JXB7 2.49E+06 6.98E−03 2.80E−9  Hu22 7.05E+064.56E−03 6.47E−10 1.7 3.80E+06 1.48E−03 4.22E−10 3.12 3.94E+06 3.47E−048.83E−11 3.23 3.53E+06 2.78E−04 7.88E−11

The human hepcidin-25 binding parameters of the mouse Mab JXB7 andcertain human engineered anti-hepcidin Mabs are shown in Table 6.Therefore, human engineered anti-hepcidin Mabs having a binding affinity(K_(D)) for human hepcidin-25 up to about 33-fold lower than that of themouse Mab JXB7 were identified. The heavy chain constant regions forMabs JXB7 and 31B2 are mouse IgG1. The heavy chain constant regions forthe other Mabs in Table 6 were human IgG4 (SEQ ID NO: 94).

TABLE 6 Binding to Human Hepcidin-25 Mab Kon (M⁻¹, s⁻¹) Koff (s⁻¹)Kinetic K_(D) (M) JXB7 3.70E+07 7.37E−03 1.99E−9  31B2 1.89E+06 1.27E−047.52E−11 Hu22 1.09E+07 8.64E−03 7.64E−10 3.23 6.20E+06 6.59E−04 9.94E−113.8 5.58E+06 7.68E−04 1.05E−10 L1.5 5.31E+06 1.82E−04 3.42E−11 3.123.68E+06 2.20E−04 5.99E−11

The cynomolgus monkey hepcidin-25 and mouse hepcidin-25 bindingparameters of various human engineered anti-hepcidin Mabs are shown inTables 7 and 8, respectively. Binding to rat hepcidin-25 wasundetectable for Mabs Hu22, 3.23, and 3.8. Generally, the Mabs Hu22 and3.23 were shown to have a K_(D) for mature cynomolgous monkey hepcidinthat was comparable that for human hepcidin-25. However, Mab 3.8 wasshown to have a K_(D) for cynomolgous monkey hepcidin-25 over 10-foldlower than for human hepcidin-25. On the other hand, Mabs Hu22, 3.23,and 3.8 were shown to have a much lower K_(D) for human hepcidin-25 thanfor mouse hepcidin-25.

TABLE 7 Binding to Cynomolgus Monkey Hepcidin-25 Mab Kon (M⁻¹, s⁻¹) Koff(s⁻¹) Kinetic K_(D) (M) Hu22 8.13E+06 8.16E−03 9.86E−10 3.23 6.51E+065.77E−04 8.88E−11 3.8 7.07E+07 6.60E−04 9.33E−12

TABLE 8 Binding to Mouse Hepcidin-25 Mab Kon (M⁻¹, s⁻¹) Koff (s⁻¹)Kinetic K_(D) (M) Hu22 1.62E+06 1.26E−01 7.76E−08 3.23 3.83E+06 1.92E−015.01E−08 3.8 3.57E+06 1.24E−01 3.46E−08

Example 3 Cell-Based Assay for Hepcidin-Induced Internalization andDegradation of Ferroportin

An in vitro cell based assay may be used to measure the neutralizationactivity of Mabs directed against human hepcidin. One in vitro cellbased assay useful to measure the neutralization activity of theantibodies of the present invention is based on hepcidin-inducedinternalization and degradation of its receptor, ferroportin. Briefly, aHEK 293 stable cell line is prepared that allows for the inducibleexpression of ferroportin (FPN). FPN is C-terminally fused with greenfluorescent protein (GFP) for tracking purposes. The inducibleexpression of the FPN-GFP molecule is controlled using the T-REx system,a commercially available tetracycline-regulated expression systemwithout viral transactivators (Invitrogen, Carlsbad, Calif.). TheFPN-GFP coding sequence is cloned into pcDNA4/TO vector, which containsan inducible promoter and a Zeocin resistance marker. The resultingconstruct is transfected into T-REx-293 cells which express theregulatory protein required for doxycycline inducible expression. Zeocinresistant clones are tested for the inducible expression of FPN-GFP.Cell growth conditions are essentially as described in themanufacturer's user manual for the T-REx System. Briefly, cells aregrown in DMEM, 10% dialyzed FBS, 20 μM FAC (ferric ammonium citrate),plus 5 μg/mL penicillin-streptomycin. Selection is maintained with 100μg/mL Zeocin and 5 μg/mL Blasticidin. Cells are plated onto 96-wellblack/clear plates that are coated with poly-D-lysine. A high resolutionfluorescent plate reader is used for reading the total fluorescence perwell.

Essentially the assay is run as follows: following trypsinization,96-well assay plate is seeded with 9,000 cells/well using theFPN-GFP/TREx 293 stable cell line. Seeding volume per well is 80 μl.Cells are allowed to attach overnight. Early the next morning, 9 μl of30 ng/mL doxycyline is added to each well to induce FPN-GFP expression.This induction is allowed occur for 8 hours. After the induction, mediais aspirated media and the wells are washed carefully with 120 μL/wellPBS. It is important to remove all liquid from each well as any leftovermedia will continue to induce expression of FPN-GFP.

The desired treatments are set up in a 96-well format for quick additionto an assay plate after washing. Final assay volume per well is 45 μL.Immediately after adding the treatments, the assay plate is read usingthe high resolution fluorescent plate reader (set at 550 volts inchannel 1). This reading is the 0 hour reading and is used to normalizefor cell number per well, which correlates with the total FLU per well.Human hepcidin-25 induced maximal internalization and degradation offerroportin at 0.5 μM. The IC₅₀ for human hepcidin-25 is approximately 8nM. For anti-hepcidin antibody neutralization assays, the humanhepcidin-25 concentration is kept at 100 nM and the anti-hepcidinantibodies are run at 2× dilutions from 0.5 μM to 8 nM. The plates areincubated for 24 hrs, after which, they are read again, and the data isgenerated as the ratio of total fluorescence units (FLU) per well at 24hours divided by the total FLU per well at 0 hours.

In this in vitro assay, human hepcidin-25 bioactivity was neutralizedwith various anti-hepcidin Mabs with an IC₅₀ measuring as shown in Table9.

TABLE 9 In Vitro Neutralization Activity of anti-Hepcidin-25 HumanEngineered Mabs Mab IC₅₀ (nM) ± Std. Error (n ≧ 4) 3.23 59.1 ± 1.2 3.1262.2 ± 5.9 3.6 54.6 ± 1.5 3.9 51.1 ± 1.8 Hu22   163 ± 12.4

Example 4 Administration of Anti-hepcidin Monoclonal Antibodies RaiseSerum Iron Levels in Cynomolgus Monkeys Treated Subcutaneously withInterleukin-6

The activity of anti-hepcidin antibodies on IL-6 induced serum irondysregulation in cynomolgus monkeys may be determined as describedbelow.

Briefly, anti-hepcidin antibodies are administered to male cynomolgusmonkeys at 1 and 10 mg/kg as an i.v. bolus. Approximately one hour afterantibody administration, the animals receive a single subcutaneousadministration of human IL-6 at 5 μg/kg. Blood samples are collected at−1 hour (prior to antibody dosing), 0 hour (immediately prior to IL-6dose) and at 1, 3, 6, 12, 24, 48, 96, 168, 336, 504, and 672 hoursfollowing IL-6 treatment. Preferably, each treatment group consists ofat least 3 animals. Serum iron levels may be measured by any methodknown in the art which is generally considered within the medicalcommunity to be an acceptable method of measuring serum iron levels.

TABLE 10 Anti-Hepcidin Mab Inhibition of IL-6 Induced Decreases in SerumIron Levels in Cynomolgus Monkeys Target Target Test/ Target Dose Dose #of Control Dose Dose Concen- Volume Group Males Articles Route Leveltration (mL/kg) 1 3 1X PBS I.V. 0 mg/mL 0 mg/mL 3.3 1X PBS S.C. 0 μg/mL0 μg/mL 1 with rHSA 2 3 1X PBS I.V. 0 mg/mL 0 mg/mL 3.3 IL-6* S.C. 5μg/mL 5 μg/mL 1 3 3 Hu22 I.V. 1 mg/mL 0.3 mg/mL 3.3 IL-6* S.C. 5 μg/mL 5μg/mL 1 4 3 Hu22 I.V. 10 mg/mL 3 mg/mL 3.3 IL-6* S.C. 5 μg/mL 5 μg/mL 15 3 3.23 I.V. 1 mg/mL 0.3 mg/mL 3.3 IL-6* S.C. 5 μg/mL 5 μg/mL 1 6 33.23 I.V. 10 mg/mL 3 mg/mL 3.3 IL-6* S.C. 5 μg/mL 5 μg/mL 1 *All IL-6doses are administered in a vehicle consisting of 1X PBS containing 0.1mg/mL recombinant human serum albumin. S.C. Subcutaneous injection;given via 1 injection site I.V. Intravenous; given as an injection viathe saphenous vein.

Compared to the PBS control group, human IL-6 treatment produces atransient reduction of serum iron levels, reaching a nadir at 12 hourspost IL-6 treatment. Intravenous administration of Hu22 and 3.23 at 10mg/kg approximately 1 hour prior to human IL-6 dose prevents the drop iniron levels due to IL-6 treatment and results in an increase in serumiron of approximately 52% and 108%, respectively, at 3 to 6 hours postIL-6 treatment. After attaining peak levels, the serum ironconcentrations subsequently decrease in these two groups and reachlevels similar to those of other groups at 24 hours. Both Hu22 (p<0.01,3 and 6 hours) and 3.23 (p<0.01, 3, 6, and 12 hours) at 10 mg/kg,produce statistically significant increases of serum iron concentrationsrelative to IL-6 group with PBS pretreatment. In contrast, neither Hu22nor 3.23 at 1 mg/kg, result in statistically significant differences inserum iron levels compared to control. Therefore, these resultsdemonstrate that the antibodies of the present invention will be usefulin treating anemia resulting from mature hepcidin bioactivity.

Example 5 Determination of Selectivity of Anti-Hepcidin Antibodies UsingMALDI-TOF

Clinical routine diagnosis of biomarkers is mostly based onimmunological, quantitative techniques—e.g., ELISA. These methods areoften not applicable for small antigens or for antigen isoforms(Sparbier, K., International Meeting of the Association of BiomolecularResource Facilities, Salt Lake City, Utah, Poster V28-S, (2008); andGutierrez, J. A., et al., (2005)).

Anti-human hepcidin Mab 31B2 is conjugated to CNBr activated sepharose 6MB resin (GE healthcare, Piscataway, N.J.) according to themanufacturer's protocol. Briefly, the sepharose resin was washed with 1mM HCl three times and the antibody was diluted into coupling buffer(100 mMNaHCO₃, 0.5 MNaCl, pH8.3). Approximately 1.7 mg of antibody wasused for conjugation at 4° C. overnight to every mg of resin. Excessantibody was washed away by 0.1 M acetate buffer pH 4. Approximately 100ml of human serum sample was incubated with 0.8 ml of thissepharose-31B2 resin at 4° C. After overnight incubation, theresin/serum mixture was packed into a column and washed with 10-20column volumes of 10 mM sodium phosphate, 0.5 M NaCl, pH 7.4. The columnwas washed with 5-10 column volumes of 10 mM sodium phosphate pH 7.4without NaCl. Finally, the column was eluted with 0.2% TFA. The elutedfractions were analyzed for molecular weights on a Voyager-DE STRinstrument (Applied Biosystems, Foster City, Calif.) in linear mode.

For molecular weights smaller than human hepcidin-25, the mass spectrumwas generated using a peptide matrix. A dominant peak corresponding tohuman hepcidin-25, (2790 Dalton) was identified. Less dominant peaks forvarious truncated forms of hepcidin, namely hepcidin-24 (2674 Dalton),hepcidin-22 (2436 Dalton), and hepcidin-20 (2192 Dalton), were alsodetectable.

For molecular weights bigger than human hepcidin-25, the mass spectrumwas generated using a protein matrix. A dominant peak corresponding tohuman hepcidin-25 (25 aa, 2790 Dalton) was still identified, but no peakcorresponding to prepro-hepcidin (84 aa, 9400 Dalton) or pro-hepcidin(60 aa, 6929 Dalton) was detectable.

Thus, immunoassays using the 31B2 Mab and human engineered versionsthereof are selective for human hepcidin-25, the active, mostphysiologically relevant form of hepcidin in human serum as compared toprecursor and/or N-terminally truncated forms thereof known to exist inhuman serum.

Example 6 Correction of Cryptic Splicing of Anti-Hepcidin-25Antibody-Encoding mRNA Upon Expression in CHO Cells

Standard molecular biology techniques may be used to prepare therecombinant expression vectors, transfect the host cells, select fortransformants, isolate host cell lines capable of expressing an antibodyof the invention, culture the host cells and recover the expressedantibodies from culture medium. Surprisingly, upon production of theMabs 3.12 and 3.23 by Chinese hamster ovary (CHO) cell suspensioncultures using a recombinant glutamine synthetase (GS) expression system(Lonza Biologics, Inc., Slough, UK), unusually high level ofantibody-protein aggregation were observed (˜15% and ˜30%, as measuredby size-exclusion chromatography, respectively). However, a high levelof aggregation was not observed when the two Mabs were transientlyexpressed in human-origin HEK-293 cells. An examination of the mRNA fromeach of the two CHO cell lines revealed the presence of transcripts ofunexpected sizes, suggesting that the nucleotide sequences encoding theantibody proteins of Mabs 3.12 and 3.23 were susceptible to crypticsplicing events. Additionally, an examination of the aggregated proteinsamples revealed the presence of truncation in the light chain protein.Sequencing of cDNA prepared from mRNA isolated from CHO cells expressingMabs 3.12 and 3.23 confirmed that the presence of cryptic introns in thelight-chain genes. The splice donor was contained in the codons encodingthe amino acid residues R/VS of LC CDR1 (amino acid 5-7 of SEQ ID NO: 43and “/” denotes the splice junction, in frame). Furthermore, a probablebranch point was found at the codons encoding the amino acid residues L1of FRL2 (SEQ ID NO: 40) with a probable poly-pyrimidine stretch in thecodons encoding the amino acids residues STL and SPL in the LCDR2 of Mab3.12 and 3.23, respectively. And lastly, the acceptor sites wereidentified at the codons encoding for the amino acid residues C/Q/Q inLCDR3 of both Mabs 3.12 and 3.23 (amino acids 1-3 of SEQ ID NO: 61; “/”denotes the probable splice junctions).

The original DNA sequences encoding the light chains of Mabs 3.12 and3.23 (SEQ ID NOs: 153 and 155, respectively) were subsequently modifiedto eliminate sequences conferring the splice donor, branch point,acceptor and poly-pyrimidine tract to the cryptic intron. Morespecifically, the donor site was changed from CGC GTA AGT to AGA GTC TCC(SEQ ID NO: 45). The branch point was changed from CTG ATC to CTC ATC(SEQ ID NO: 162); the poly pyrimidine tract from TCC ACC CTG to AGC ACACTG (SEQ ID NO: 77) and from TCC CCC CTG to AGC CCA CTG (SEQ ID NO: 78)in 3.12 and 3.23, respectively; and the acceptors from TGT CAG CAG TGGto TGC CAA CAA TGG (SEQ ID NO: 100).

Accordingly, subsequent expression of the light chains of Mabs 3.12 and3.23 was effectuated by recombinant expression vectors harboring themodified DNA sequences as shown in SEQ ID NOs: 12 and 13, respectively.The amount of aggregated antibody produced upon expression of themodified nucleic acid sequences in CHO cells was determined to be 1% orless for both the 3.12 and 3.23 Mabs.

Modifications to the DNA sequences encoding the light chains of variousother antibodies of the present invention would likewise be expected tobe of great benefit since the splice donor, poly pyrimidine tract, andthe splice acceptors are from the codon encoding LCDRs that givespecificity to the antibody. The branch point is in FRL2 and the LLIYsequence is highly conserved in light chain frameworks. Moreover, the QQmotif, whose encoding nucleotide sequence spans the identified spliceacceptor region, is conserved within the LCDR3 of many of theanti-hepcidin mAbs of the invention. Generally, many light chaingermline sequences contain one or both of these Q codons (CAG) that canserve as the acceptor of potential cryptic introns.

1. An antibody that comprises six CDRs selected from the group consisting of: (i) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs: 41, 53, 31, 63, 84, and 46, respectively; (ii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs: 43, 30, 31, 32, 44, and 46, respectively; (iii) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs: 43, 53, 61, 63, 85, and 46, respectively; and (iv) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 having the amino acid sequences as shown in SEQ ID NOs: 43, 57, 61, 63, 84, and 46, respectively.
 2. The antibody of claim 1 that comprises a heavy chain variable region and a light chain variable region wherein the amino acid sequence of the heavy chain comprises the amino acid sequences shown in SEQ ID NO: 151 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:
 125. 3. The antibody of claim 1 that comprises a heavy chain variable region and a light chain variable region wherein the amino acid sequence of the heavy chain comprises the amino acid sequences shown in SEQ ID NO: 150 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:
 124. 4. The antibody of claim 1 that consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 8 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 16. 5. The antibody of claim 1 that consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 9 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 17. 6. A polynucleotide comprising a nucleotide sequence that encodes an amino acid sequence as shown in SEQ ID NOs: 124, 125, 126, 127, 148, 150, 151, or
 128. 7. The polynucleotide according to claim 6 wherein the nucleotide sequence encodes an amino acid sequence as shown in SEQ ID NOs: 8, 9, 16, or
 17. 8. The polynucleotide according to claim 7 wherein the nucleotide sequence encodes an amino acid sequence as shown in SEQ ID NO: 8 and an amino acid sequence as shown in SEQ ID NO:
 16. 9. The polynucleotide according to claim 7 wherein the nucleotide sequence encodes an amino acid sequence as shown in SEQ ID NO: 9 and an amino acid sequence as shown in SEQ ID NO:
 17. 10. The polynucleotide according to claim 8 wherein the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 16 is as shown in SEQ ID NO:
 12. 11. The polynucleotide according to claim 6 wherein the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 17 is as shown in SEQ ID NO:
 13. 12. A recombinant expression vector comprising a polynucleotide according to claim
 10. 13. A recombinant expression vector comprising a polynucleotide according to claim
 11. 14. A host cell which has been transformed by a vector according to claim
 12. 15. A host cell which has been transformed by a vector according to claim
 13. 16. The host cell according to claim 14 which is a CHO cell.
 17. The host cell according to claim 15 which is a CHO cell.
 18. A method of increasing serum iron levels, reticulocyte count, red blood cell count, hemoglobin, and/or hematocrit comprising administering to a subject an effective amount of the antibody of claim
 1. 19. A method of treating anemia in a subject, comprising administering to the subject an effective amount of the antibody of claim
 1. 20. The method of claim 19 wherein the antibody consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 8 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 16. 21. The method of claim 19 wherein the antibody consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 9 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 17. 22. A process for producing an antibody comprising the steps of: (i) culturing a host cell according to claim 16 under conditions suitable to allow expression of said antibody; and (ii) recovering the expressed antibody.
 23. A process for producing an antibody comprising the steps of: (i) culturing a host cell according to claim 17 under conditions suitable to allow expression of said antibody; and (ii) recovering the expressed antibody.
 24. An antibody obtained by the process according to claim
 22. 25. An antibody obtained by the process according to claim
 23. 26. A pharmaceutical composition comprising the antibody of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.
 27. A pharmaceutical composition comprising the antibody of claim 4, and a pharmaceutically acceptable carrier, diluent, or excipient.
 28. An immunoassay comprising a) obtaining a sample to be assayed for human mature hepcidin; b) contacting the sample with an antibody of claim 1 under suitable conditions for antibody binding and allowing any human mature hepcidin present to form a complex with the antibody; and c) detecting the presence or absence of the complex; and/or determining the amount of the complex in the sample by an immunodetection method.
 29. The immunoassay of claim 28 wherein the antibody consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 8 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 16. 30. The immunoassay of claim 28 wherein the antibody consists of two heavy chains and two light chains, wherein the amino acid sequence of each of the heavy chains is as shown in SEQ ID NO: 9 and the amino acid sequence of each of the light chains is as shown in SEQ ID NO:
 17. 